Name: xenograft skin model to manipulate human immune responses in vivo
Uploaded: 2022-06-29T13:30:00
Description: Watch this Scientific Journal Video about Xenograft Skin Model to Manipulate Human Immune Responses In Vivo at JoVE.com

This work was funded in part by sponsored research agreements from TRex Bio and grants from the NIH (1R01AR075864-01A1). JMM is supported by the Cancer Research Society (grant 26005). We acknowledge the Parnassus Flow Cytometry Core supported in part by grants NIH P30 DK063720, S10 1S10OD021822-01, and S10 1S10OD018040-01.

The present study was approved and performed in compliance with UCSF IACUC (AN191105-01H) and IRB (13-11307) protocols. Skin samples, discarded as part of routine elective surgical procedures, such as hernia repair, were used for the present research. The skin samples are either de-identified and certified as Not Human Subjects Research or, if clinically identifying information is required for downstream analyses, patients provided written consent under IRB protocol 13-11307. No other inclusion or exclusion criteria were...

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The mouse xenograft skin transplant model is a key technique to mechanistically dissect human skin immune responses in an in vivo setting14. Successful skin xenograft transplants rely upon appropriate preparation of mice and&#160;skin specimens and mice and adherence to aseptic rodent surgery methods15. Rapid cooling and proper storage of skin samples at cold temperatures in media (such as sterile saline) are important to ensure continued tissue health prior to tra...

Mouse models are frequently used to make inferences about human biology and disease, partly due to their experimental reproducibility and capacity for genetic manipulation. However, mouse physiology does not completely recapitulate human organ systems, particularly skin, and therefore has limitations for use as a preclinical model in drug development1. Anatomical differences between mouse and human skin include differences in epithelial thicknesses and architecture, lack of murine eccrine sweat glands, and variations in hair cycling2. Furthermore, both the innate and adaptive arms of the immune system are divergent between the two species3. Mouse skin contains a unique immune population of dendritic epidermal T cells (DETCs), has a higher abundance of dermal &#947;&#948; T cells, and varies in immune cell subset localization in comparison to human tissue4. Therefore, experimental findings regarding human skin biology and inflammation benefit from validation with human tissue. While in vitro and organoid culture systems are widely utilized tools to study human tissue, these systems are limited by absent or incomplete immune reconstitution and a lack of connection to peripheral vasculature5. The humanized xenograft skin transplant model aims to allow for therapeutic or biological manipulation of immune and non-immune pathways in human tissues in vivo.
The human skin xenograft model has been utilized to study skin physiology and pharmacology, analyze immune rejection and responses, dissect human skin cancer mechanisms, and understand skin diseases and wound healing6. While applicable to multiple fields of skin research, the xenograft model has lower throughput than in vitro studies and lacks the ease of genetic manipulation employed in mouse models. Time points within this model may range from weeks to months, and successful grafting requires appropriate facilities and equipment to perform these surgeries. However, the xenograft model supplies biological and physiological context to experiments, while organoid culture systems, such as tissue explants, often require replicating a myriad of moving parts, such as exogenous signals, at specific time intervals7. Therefore, this model is best utilized to further validate findings observed in vitro and within mouse models, or for work that is not otherwise biologically feasible. Appropriate use of the xenograft model provides a unique opportunity to study and manipulate intact human tissue in vivo.
Optimization of the xenograft skin transplant model has relied on decades of research to preserve graft integrity over time. Critical to this process is utilizing the non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mouse, which lacks B and T adaptive immune cells, functional NK cells, and has deficiencies in macrophage and dendritic cells8. The immunodeficient nature of these NSG hosts allows for the transplant of human hematopoietic cells, patient-derived cancers, and skin8,9,10. Despite this immunosuppressive host environment, additional suppression of mouse neutrophilic immune responses by anti-GR1 administration is necessary for graft success10. The major roadblocks in transplanting intact tissue are infection, rejection, and difficulty in re-establishing blood flow to the graft, sometimes leading to loss of dermal and epidermal integrity11. Techniques including administration of anti-FR1 and use of appropriate graft depth improve graft survival10. Meticulous optimization makes it possible to perform human xenograft skin transplants on NSG mice with high efficiency and survival rates, ranging from 90%-100%.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Fisher</td>
			<td>SF100-20</td>
			<td>Fixative for histology</td>
		</tr>
		<tr>
			<td>3M Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>surgical glue</td>
		</tr>
		<tr>
			<td>Alexa 700 CD45 monoclonal antibody (Clone 30F11)</td>
			<td>Thermo Fischer</td>
			<td>56-0451-82</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Anti-GR1 clone RB6-8C5</td>
			<td>BioXcell</td>
			<td>BE0075</td>
			<td>Anti-rejection</td>
		</tr>
		<tr>
			<td>APC mouse anti-human CD25&#160; (Clone 2A3)</td>
			<td>BD Biosciences</td>
			<td>340939</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>APC-eFluor 780 anti-human HLA-DR (Clone LN3)</td>
			<td>eBioscience</td>
			<td>47-9956-42</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Autoclave pouches</td>
			<td>VWR&#160;</td>
			<td>89140-800</td>
			<td>For autoclaving tools and paper towels</td>
		</tr>
		<tr>
			<td>Brilliant Violet 60 anti-human CD4 antibody (Clone OKT4</td>
			<td>Biolegend</td>
			<td>317438</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Brilliant Violet 65 anti-human CD8a antibody (Clone RPA-T8)</td>
			<td>Biolegend</td>
			<td>301042</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Brilliant Violet 711 anti-human CD3 antibody (Clone OKT3)</td>
			<td>Biolegend</td>
			<td>317328</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Buprenex 0.3 mg/mL</td>
			<td>Covetrus</td>
			<td>059122</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Carprofen 50 mg/mL</td>
			<td>Zoetis</td>
			<td>NADA # 141-199</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>4188</td>
			<td>Skin digestion</td>
		</tr>
		<tr>
			<td>D42 Dermatome blade</td>
			<td>Humeca</td>
			<td>5.D42BL10</td>
			<td>dermatome (1 blade per sample)</td>
		</tr>
		<tr>
			<td>Dermatome D42</td>
			<td>Humeca</td>
			<td>4.D42</td>
			<td>dermatome</td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>Bard-Parker</td>
			<td>371610</td>
			<td>skin preparation</td>
		</tr>
		<tr>
			<td>Dissecting T-Pins; 1-1/2 inch, 1000/CS 1.5</td>
			<td>Cole-Parmer</td>
			<td>UX-10915-03</td>
			<td>To pin skin specimen for dermatome</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>medicon</td>
			<td>02.04.10</td>
			<td>sample preparation and mouse dissection</td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma-Aldrich</td>
			<td>DN25-1G</td>
			<td>Skin digestion</td>
		</tr>
		<tr>
			<td>eBioscience Foxp3 / Transcription Factor Fixation/Permeabilization Concentrate and Diluent</td>
			<td>eBioscience</td>
			<td>00-5521-00</td>
			<td>Flow cytometry analysis: Cell Fixation and Permeabilization</td>
		</tr>
		<tr>
			<td>eFluor-450 FOXP3 monoclonal antibody (Clone PCH101)</td>
			<td>eBioscience</td>
			<td>48-4776-42</td>
			<td>Flow cytometry analysis: Intracellular protein staining</td>
		</tr>
		<tr>
			<td>Electric clippers</td>
			<td>Kent</td>
			<td>CL8787-KIT</td>
			<td>hair removal</td>
		</tr>
		<tr>
			<td>Epredia&#160;Shandon Instant Eosin</td>
			<td>Fisher Scientific</td>
			<td>6765040</td>
			<td>H&#38;E</td>
		</tr>
		<tr>
			<td>Epredia&#160;Shandon Instant Hematoxylin</td>
			<td>Fisher Scientific</td>
			<td>6765015</td>
			<td>H&#38;E</td>
		</tr>
		<tr>
			<td>FITC anti-human CD45 (Clone HI30)</td>
			<td>Tonbo Biosciences</td>
			<td>35-0459-T100</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Forceps&#160;</td>
			<td>medicon</td>
			<td>07.60.07</td>
			<td>sample preparation and mouse dissection</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Fisherbrand</td>
			<td>22-362-178</td>
			<td>Sample preparation</td>
		</tr>
		<tr>
			<td>Heating lamp</td>
			<td>Morganville Scientific</td>
			<td>HL0100</td>
			<td>Post-surgical care</td>
		</tr>
		<tr>
			<td>Heating pads 4&#34; x 10&#34;</td>
			<td>Pristech</td>
			<td>20415</td>
			<td>Surgical heat supply</td>
		</tr>
		<tr>
			<td>Insulin 1cc 12.7 mm syringes</td>
			<td>BD</td>
			<td>329410</td>
			<td>drug administration</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>United States Pharmacopeia (USP)&#160;</td>
			<td>NDC 66794-013-25</td>
			<td>Anesthesia&#160;</td>
		</tr>
		<tr>
			<td>Isoflurane machine</td>
			<td>VetEquip</td>
			<td>911103</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Nair for Men</td>
			<td>Nair</td>
			<td>&#8206; 10022600588556</td>
			<td>hair removal</td>
		</tr>
		<tr>
			<td>Neomycin and Polymyxin Bisulfates and Bacitracin Zinc Ophthalmic ointment</td>
			<td>Dechra</td>
			<td>&#160;NDC 17478-235-35</td>
			<td>eye ointment to prevent drying</td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice</td>
			<td>The Jackson Laboratory</td>
			<td>005557</td>
			<td>Mice</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>Kleenex</td>
			<td>100848</td>
			<td>May be autoclaved for sterile surfaces</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td>Semitransparent sealing film</td>
		</tr>
		<tr>
			<td>PE mouse anti-human CD127 (Clone HIL-7R-M21)</td>
			<td>BD Biosciences</td>
			<td>557938</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>PE-Cy-7 mouse anti-Ki-67 (Clone B56)</td>
			<td>BD Biosciences</td>
			<td>561283</td>
			<td>Flow cytometry analysis: Intracellular protein staining</td>
		</tr>
		<tr>
			<td>PerCP-eFluor-710 CD152 (CTLA-4) monoclonal antibody (Clone 14D3)</td>
			<td>eBioscience</td>
			<td>46-1529-42</td>
			<td>Flow cytometry analysis: Intracellular protein staining</td>
		</tr>
		<tr>
			<td>Permeabilization Buffer 10x</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td>Flow cytometry analysis: Intracellular protein staining buffer</td>
		</tr>
		<tr>
			<td>Petri Dish 150 mm</td>
			<td>Corning</td>
			<td>430597</td>
			<td>Sample storage</td>
		</tr>
		<tr>
			<td>Plastic Wrap</td>
			<td>Fisherbrand</td>
			<td>22-305-654</td>
			<td>Site preparation</td>
		</tr>
		<tr>
			<td>Providone-Iodine Swab stick</td>
			<td>PDI</td>
			<td>S41350</td>
			<td>Site sterilization</td>
		</tr>
		<tr>
			<td>Soft-Feed and Oral Hydration (Napa Nectar)</td>
			<td>Se Lab Group Inc</td>
			<td>NC9066511&#160;</td>
			<td>For supplementing poorly recovering mice post-surgery</td>
		</tr>
		<tr>
			<td>Specimen Collection Cups</td>
			<td>Fisher Scientific</td>
			<td>22-150-266</td>
			<td>sample storage</td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pad</td>
			<td>Fisherbrand</td>
			<td>22-363-750</td>
			<td>skin preparation</td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Media for sample storage</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Hospira</td>
			<td>NDC 0409-4888-02</td>
			<td>For drug dilution</td>
		</tr>
		<tr>
			<td>Tegaderm Film 4&#8221; x 43/4&#8221;&#160;</td>
			<td>3M</td>
			<td>1626</td>
			<td>transparent film wound dressing</td>
		</tr>
		<tr>
			<td>Vaseline Petrolatum Gauze 3&#8221; x 8&#8221;&#160;</td>
			<td>Kendall</td>
			<td>414600</td>
			<td>wound dressing</td>
		</tr>
		<tr>
			<td>Violet 510 Ghost Dye&#160;</td>
			<td>Tonbo Biosciences</td>
			<td>13-0870-T100</td>
			<td>Flow cytometry analysis: Viability dye</td>
		</tr>
	</tbody>
</table>

xenograft skin model to manipulate human immune responses in vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Human skin xenografts were performed on NSG mice inside a super-barrier animal facility. Success was defined by the prolonged graft and mouse survival and behavioral health of mice post-transplant. Poor survival during the week following surgery was initially observed as the biggest barrier to experimental success, with up to 50% of mice requiring euthanasia. Improving sterile technique and better support of mouse body temperatures during and immediately after surgery increased surgical survival consistently to over 80% ...

Watch this Scientific Journal Video about Xenograft Skin Model to Manipulate Human Immune Responses In Vivo at JoVE.com

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

We outline techniques necessary for the human xenograft skin transplant model, an important tool for the manipulation of immune and non-immune pathways in human tissues in vivo. This technique better recapitulates human skin physiology than in vitro methods, allowing for preclinical experimental manipulation in vivo. Xenograft skin transplants can be used for preclinical testing of compounds targeting human skin inflammation in vivo.Bandaging mice can be particularly challenging, so we would advise practicing the bandaging process on healthy anesthetized mice prior to performing surgeries. To begin, prepare to dermatome the human skin sample in a sterilized negative pressure tissue culture hood on a sterilized dissection board. Place the skin sample with epidermis side up on the dissection board and wipe the epidermis with a sterile alcohol prep pad followed by PBS.Now use a 1.5 inch dissecting T pin to fix the closer edge of the skin. Then dermatome the skin specimen at 400 micrometer thickness and apply steady pressure while cutting forward at an angle of 30 to 45 degrees. Place a sterile gauze soaked in sterile PBS at the bottom of the Petri dish and place the skin with epidermis side up onto the wet gauze.Seal and cover the plate edges with a semi-transparent sealing film to ensure the sample is not contaminated and store the sample at four degrees Celsius before grafting. Prepare the sterile instruments and surgical station for grafting and use autoclaved paper towels as sterile surfaces for instrument and mouse placement. After anesthesia induction, transfer the mouse to a heating pad or another heat source and administer the ophthalmic ointment by dabbing a small drop of ointment on the eye with a gloved finger.Pinch the skin and subcutaneously inject analgesics at an angle parallel to the body. After lifting the mouse by the tail, expose the abdomen and inject 100 micrograms per 100 microliters of anti-Gr1 intraperitoneally at a 30 degree angle using a one milliliter insulin syringe. Then use animal safe electric clippers to shave the middle and upper portions of the dorsal side of the mouse.After clearing all the hairs, apply a generous amount of hair removal ointment onto the shaved skin. Wait for 30 seconds to one minute, then wipe the hair removal ointment completely with a paper towel and PBS. After transferring the mouse to the surgical station, sterilize the surgical area and place a sterile plastic wrap over the mouse.Cut a window in the plastic slightly larger than the size of the area to be grafted. Hold the donor skin firmly in place with the forceps backside and cut a rectangle shaped 10 by 10 millimeter portion of donor skin to be grafted alongside the forceps with a scalpel. Pull the skin away from the body to avoid cutting deeply into the fascia.To create a graft bed, snip a rectangular area of mouse skin matching the size of the donor skin piece. Then place the donor skin piece with epidermis side up onto the prepared graft bed. Using the back of the forceps, manipulate the skin sliding back and forth until the donor skin lies completely flat against the graft bed.Add surgical glue tissue adhesive drops where the donor skin meets the mouse skin and hold the mouse and donor skin together with forceps for one to two seconds so that the glue adheres to the tissues. Completely seal the graft's edge and allow the glue to dry thoroughly. Next, cut petrolatum gauze large enough to cover the graft area completely.Cover the graft with the petrolatum gauze and lightly press the gauze against the skin using forceps. Cut a strip of a transparent film dressing lengthwise so that the width is large enough to cover the mouse's wound. Firmly press the transparent film dressing with the adhesive side down over the gauze.Quickly roll the mouse to wrap the dressing completely around the torso, ensuring it fits tightly without impeding respiration and all limbs are free for movement. After placing the mouse in a recovery cage, monitor it until it is alert and moving around. Provide a heat source on the part of the cage for at least 15 minutes following recovery.Analysis of grafts 10 to 21 days post-transplant showed that the graft remained adherent around the edges where the donor's skin meets the mouse's skin and the graft remained thicker and more inflamed than healthy human skin. Analysis by flow cytometry revealed the sustained presence of human immune cells within most grafts, but counts varied between xenografts. The successful graft maintained the human immune cells.However, despite continued adherence and survival of donor tissue, grafts which failed to maintain the human immune cells are deemed unsuccessful. Analysis of hematoxylin and eosin stained sections of the graft after 35 days showed intact epidermis and the dermis is moderately cellular composed of plump oval fibroblasts with pale syncytial cytoplasm and scattered lymphocytes. Analysis of the graft after 50 days showed similar results with intact epidermis and the dermis containing oval fibroblasts and slightly enhanced extracellular matrix deposition.Successful skin xenografts rely upon appropriate preparation of skin specimens, suppression of mouse neutrophilic immune responses by anti-Gr1 administration, adherence to aseptic rodent surgery methods, and secure bandaging of mice. The human xenograft skin transplant model allows for researchers to study human immune responses in the intact organ in vivo, providing a new avenue for translational research.

Research

JoVE Journal

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