Name: murine full-thickness skin transplantation
Uploaded: 2017-01-02T15:00:00.000+00:00
Duration: 7 min 59 s
Description: Watch this Scientific Journal Video about Murine Full-thickness Skin Transplantation at JoVE.com

This work was funded by NIH grant R01AI077610.

All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health (NIH) and were approved by the Johns Hopkins University Animal Care and Use Committee (JHUACUC). The specific procedures were performed under the approved ACUC protocols MO13M292 and MO13M370.
1. Donor Skin Harvest
<ol>
	<li>Anesthetize the donor mouse with isoflurane (induction vaporizer at 4%, maintenance at 1 - 2% through the mouse cone). Use the toe pinch withdrawal reflex to monitor the depth of anesthesia.</li>
	<li>Using an electric razor, shave the back of the animal and disinfect it with 10% povidone iodine (e.g., Betadine).</li>
	<li>Using sterile scissors, gloves, and aseptic technique, harvest donor back skin from the hip to the neck, with blunt dissection at the level of the areolar connective tissue.</li>
	<li>Euthanize the animal by cervical dislocation after harvesting the skin graft.</li>
	<li>Under a microscope, separate the connective tissue, fat tissue, and panniculus carnosus from the back skin using fine tenotomy scissors. The panniculus carnosus is a thin, transparent muscle responsible for skin twitching movements.</li>
	<li>Using sterile instruments technique, cut out 15-mm x 15-mm grafts from the back skin for a 10-mm x 10-mm to 15-mm x 15-mm graft bed.</li>
	<li>Store the grafts on gauzes soaked with sterile phosphate-buffered saline (PBS) in a petri dish on ice. 
	NOTE: 8 - 10 grafts can be obtained from one donor mouse.</li>
</ol>
2. Recipient Skin Transplant
<ol>
	<li>Anesthetize the recipient mouse with isoflurane (induction vaporizer at 4%, maintenance at 1 - 2% through the mouse cone). Use the toe pinch withdrawal reflex to monitor the depth of anesthesia.</li>
	<li>Administer 0.02 mg/kg BW of Buprenorphine for postoperative pain relief.</li>
	<li>Shave the side of the back of the animal where the graft will be inserted and disinfect with 10% povidone iodine.</li>
	<li>Using scissors, cut a 10-mm x 10-mm to 15-mm x 15-mm square of skin. The defect size should be slightly larger (10%) than the graft. Cut as superficially as possible. Take care to preserve the panniculus carnosus and vessels. The panniculus carnosus can be distinguished from the underlying fascia by its mobility and the blood vessels that run superficial to it.</li>
	<li>Position the graft on the graft bed, avoiding folds along the edges.</li>
	<li>Place 8 sutures on the corners and on the middle of each edge. For each suture, pass the needle through the graft and then through the panniculus carnosus of the graft bed below the surrounding recipient skin.</li>
	<li>Remove the anesthetic mask and let the animal recover partially from anesthesia before applying the adhesive bandage.</li>
	<li>Wrap the recipient mouse in an adhesive bandage with folded gauze over the graft. Make the bandage by combining two bandages, cutting the adhesive part of one and placing the two absorbent pads together.</li>
	<li>Monitor the mouse closely during recovery to ensure that the bandage is not restricting thorax excursion and breathing. Remove the bandage if the respiratory rate decreases or the animal starts to gasp or breath shallowly.</li>
</ol>
3. Postoperative Care
<ol>
	<li>Administer enrofloxacin 5 mg/kg after surgery for infection prophylaxis.</li>
	<li>Place the transplanted mouse in a clean cage over a microwavable heating pad&#160;until it fully recovers from anesthesia.</li>
	<li>Observe the mouse for 1 hr postoperatively prior to returning it to the housing facility.</li>
	<li>Seven days after surgery, anesthetize the mouse as in Step 2.1. Remove the bandage by cutting only through the ventral side of the bandage.</li>
	<li>Observe and palpate the graft on the following day for signs of scabbing, contraction, or hardness. If present, the graft may not have achieved proper vascularization and should be considered a technical failure.</li>
	<li>Monitor daily for signs of rejection. Consider grafts rejected when &#8805;90% of the graft tissue becomes necrotic.</li>
	<li>Euthanize the rejected animals and harvest them for analysis.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+Hind+Limb+Transplantation+in+the+Mouse.">Orthotopic Hind Limb Transplantation in the Mouse.</a> J Vis Exp. , (2016).</li><li>Oh, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Microsurgical+Model+for+Heterotopic,+En+Bloc+Chest+Wall,+Thymus,+and+Heart+Transplantation+in+Mice.">A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice.</a> J Vis Exp. , (2016).</li><li>Oberhuber, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+cervical+heart+transplantation+model+using+a+modified+cuff+technique.">Murine cervical heart transplantation model using a modified cuff technique.</a> J Vis Exp. , e50753 (2014).</li><li>Jones, T. R., Shirasugi, N., Adams, A. B., Pearson, T. C., Larsen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+identifies+selectins+that+regulate+T+cell+traffic+into+allografts.">Intravital microscopy identifies selectins that regulate T cell traffic into allografts.</a> J Clin Invest. 112, 1714-1723 (2003).</li><li>Celli, S., Albert, M. L., Bousso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+innate+and+adaptive+immune+responses+underlying+allograft+rejection+by+two-photon+microscopy.">Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy.</a> Nat Med. 17, 744-749 (2011).</li><li>Medawar, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+and+fate+of+skin+autografts+and+skin+homografts+in+rabbits:+A+report+to+the+War+Wounds+Committee+of+the+Medical+Research+Council.">The behaviour and fate of skin autografts and skin homografts in rabbits: A report to the War Wounds Committee of the Medical Research Council.</a> J Anat. 78, 176-199 (1944).</li><li>Billingham, R. E., Brent, L., Medawar, P. B., Sparrow, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+studies+on+tissue+transplantation+immunity.+I.+The+survival+times+of+skin+homografts+exchanged+between+members+of+different+inbred+strains+of+mice.">Quantitative studies on tissue transplantation immunity. I. The survival times of skin homografts exchanged between members of different inbred strains of mice.</a> Proc R Soc Lond B Biol Sci. 143, 43-58 (1954).</li><li>Garrod, K. R., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+skin+transplantation.">Murine skin transplantation.</a> J Vis Exp. , (2008).</li><li>Schmaler, M., Broggi, M. A., Rossi, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+tail+skin+to+study+allogeneic+CD4+T+cell+responses+in+mice.">Transplantation of tail skin to study allogeneic CD4 T cell responses in mice.</a> J Vis Exp. , e51724 (2014).</li><li>Bergstresser, P. R., Toews, G. B., Gilliam, J. N., Streilein, J. 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The authors have nothing to disclose.

Since its introduction by Medawar, first in human studies and then in rabbits and mice, skin transplantation has been an invaluable model for the study of allogeneic immune responses6,7. In this manuscript, we present a model of large-scale, non-vascularized, full-thickness skin transplantation using the upper and lower back skin. Various alternative methods, including using the tail skin or ear skin of the mouse as the graft tissue source, have been reported to date8,9. These models present the distinct advantage of technical simplicity and allow for high-volume throughput. The relative deficiency of epidermal Langerhans cells and dermal dendritic cells in tail skin grafts, however, leads to delayed rejection as well as to facilitated graft acceptance by the host, particularly in minor MHC mismatch models10,11. Therefore, full-thickness trunk skin grafting is considered a more robust model and more suitable to study T-cell-mediated acute rejection12.
In addition, our model employs simple techniques and does not require advanced microsurgical skills. The operating time is short (~ 10 min per mouse from the onset of anesthesia to the completion of the procedure), and the postoperative morbidity and mortality is negligible. The overall operative mortality (death within 24 hr after surgery plus death before removal of the bandage) has been reported to be 3.8%13. Most of the complications arise from anesthesia overdose or overly-restrictive bandages, which can be easily prevented by close monitoring of respiratory function during the immediate postoperative period and by rapid removal of the bandage at the earliest signs of respiratory distress6,17.
The most critical factor in the process of engraftment is the revascularization of graft tissue by the ingrowth of host vessels to the graft6. The harvested skin requires careful preparation, and the dissection of the panniculus carnosus is a critical step. We prefer to remove the panniculus carnosus under a microscope. It is recommended to start the dissection from one of the edges and to advance slowly by pushing the muscle and separating it from the skin with the blade of a curved tenotomy angled slightly. The site of the graft bed should be far from joints and the spine. Before making incisions into the recipient mouse, the shoulder and hip should be moved to visualize the skin folds that help to demarcate the level of the joints. When removing the recipient skin, it is important to avoid damaging the blood vessels that run superficial to the panniculus carnosus. If bleeding occurs, apply local compression and remove blood and clots that may interfere with engraftment. Finally, the bandage should not cause breathing difficulties that compromise recovery, although it must be sufficiently constrictive to prevent displacement of the graft and auto-mutilation during the process of revascularization. Plastic Strips Band-Aids are adherent and strong enough for this purpose. We combine two bandages to completely wrap the mouse waist and to allow the placement of a folded gauze between the bandage and the graft (Figure 1). The bandage is removed by cutting the ventral side of it, and the rest is removed by the animal, in order to avoid disrupting the graft while trying to remove the bandage manually.
In summary, full-thickness skin transplantation is a well-established model with abundant references in the literature. The procedure is easy to perform, the operating time is short, and the postoperative complications are limited. The critical steps are related to meticulous donor skin harvest and the correct placement of the recipient bandage. Future studies involving biomaterials that promote angiogenesis and tissue repair may further improve the success rate of the procedure.

Organ transplantation is the treatment of choice for patients with end-stage organ failure, and outcomes have improved remarkably with advances in surgical procedures and immunosuppression protocols. However, long-term immunosuppression is associated with significant side effects, and the development of new strategies that promote tolerance remains the goal of modern transplantation research.
Numerous animal models have been developed for basic research in transplantation, to study the mechanisms of allograft rejection and to test immunosuppression approaches for preventing graft rejection and for promoting long-term tolerance1-3. Mouse models have become the mainstay of immunological research due to the exclusive and vast availability of diagnostic and therapeutic antibodies and well-defined inbred and transgenic strains. Skin transplantation is a simple procedure that does not require special microsurgical skills and can be easily monitored postoperatively. Taken together, mouse skin transplantation has been an exceptional tool to study many aspects involved in the alloimmune response, including antigen delivery, cell trafficking, and tissue destruction during graft rejection4,5.
Here, we show the step-by-step procedure for full-thickness skin transplantation using the mouse model, and we describe the important steps necessary for a successful engraftment of the transplanted skin.

<table><tbody><tr><td>Straight micro forceps</td><td>Sigma</td><td>F4017</td><td></td></tr><tr><td>Curved micro forceps</td><td>Aesculap</td><td>BD333R</td><td></td></tr><tr><td>Curved Stevens tenotomy scissors</td><td>Aesculap</td><td>BC905R</td><td></td></tr><tr><td>Mayo dissecting scissors</td><td>Sigma</td><td>S3146</td><td></td></tr><tr><td>Micro needle holder</td><td>Aesculap</td><td>BM563R</td><td></td></tr><tr><td>Sterile gauze</td><td>Covidien</td><td>441218</td><td></td></tr><tr><td>6-0 Nylon suture</td><td>MWI</td><td>31849</td><td></td></tr><tr><td>Plastic Strips Band&amp;#45;Aid</td><td>Johnson &amp;amp; Johnson</td><td></td><td>Obtained from pharmacy</td></tr><tr><td>10 cm Petri dish</td><td>Fisherbrand</td><td>FB0875712</td><td></td></tr><tr><td>PBS</td><td>Quality Biological</td><td>119069131</td><td></td></tr><tr><td>Buprenorphine</td><td></td><td></td><td>DEA Number required; Obtained from hospital pharmacy</td></tr><tr><td>Enrofloxacin</td><td>Bayer Health Care</td><td>186599</td><td></td></tr></tbody></table>

murine full-thickness skin transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

The placement of the bandage on the recipient mouse is an important step of the procedure. The skin graft is positioned on the recipient trunk, between the shoulder, hip, and spine (Figure 1). The bandage is made with folded gauze and the combination of two plastic adhesive bandages. The recipient mouse is placed with the graft down over the gauze on the center of the bandage. Using two curved micro forceps, the lower end of the bandage is pulled first, and then the top of the bandage is wrapped over the mouse to the abdomen.
In complete mismatch models, full-thickness skin grafts are usually rejected in 8 to 12 days. In minor mismatch models, rejection responses are slower, more variable, and the skin graft appearance is characterized by less distinct changes, such as contraction or loss of hair (Figure 2). Acute graft rejection generally begins with swelling and erythema of the graft. These events are followed by graft desiccation, shrinkage, and scab formation (Figure 2B and C). Depending on the degree of MHC mismatch and the immunosuppression protocol, rejection can occur by a subacute process, marked by subtle changes such as loss of hair, pigmentation, dermal ridges, and graft volume. In these cases, a rejected graft appears shiny, white, and hairless, with uneven edges (Figure 2F)14.
Utilizing this model of skin transplantation, we investigated a novel approach targeting T-cell metabolism to prevent graft rejection15. It is well known that metabolic signaling pathways play critical roles in dictating the outcomes of T-cell responses16. Naive T cells rely on mitochondrial oxidative phosphorylation to generate the energy necessary for basic immune surveillance. However, upon activation, effector T cells metabolically reprogram to aerobic glycolysis and exhibit increased glutamine metabolism16,17. Using Balb/c as donors and C57BL/6 as recipients, we observed that inhibiting glycolysis and oxidative phosphorylation (2DG+metformin) or glutamine metabolism alone (DON) prolonged skin graft survival, but blocking the three pathways simultaneously resulted in significantly-increased graft survival (Figure 3C). Moreover, triple metabolic therapy was more effective than conventional cyclosporine or rapamycin (Figure 3A and B). Permanent acceptance was not achieved, and further strategies, including depletion therapy, costimulation blockade, or newer metabolic therapies, may promote better induction of transplant tolerance. However, the study provided two novel insights: the use of non-specific inhibitors to target selective immune cells with increased metabolic demands, as well as the requirement to block the three metabolic pathways simultaneously to obtain more robust immunosuppression in graft rejection.
Calcineurin inhibitors and mammalian target of rapamycin (mTOR) inhibitors constitute most of the conventional immunosuppressive therapy used in clinical transplantation. Median graft survival rates of Balb/c to C57BL/6 skin transplants treated with cyclosporine and rapamycin were 17 and 16 days, respectively. In comparison, recipients treated with metabolic inhibitors that inhibit T cell glycolysis, mitochondrial oxidative phosphorylation, and glutamine metabolism had prolonged survival: the median graft survival was 29 days. Furthermore, the histology of skin grafts from the metabolic therapy group exhibited more intact tissue alignment and less lymphocytic inflammatory infiltrate (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/55105/55105fig1.jpg" /> 
Figure 1: Covering of the Graft with the Bandage. (A) Skin graft placed on the right trunk. (B) Bandage made with a folded gauze and two plastic strips bandages. (C) Recipient mouse placed on the gauze. (D-F) Bandage wrapped around the mouse. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55105/55105fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55105/55105fig2.jpg" /> 
Figure 2: Skin Graft Appearance during Acute and Chronic Rejection. (A) Balb/c to C57BL/6 (complete mismatch) full-thickness skin transplantation without evidence of rejection (day 8). (B) Balb/c to C57BL/6 full-thickness skin transplantation with 50% graft rejection (day 13). (C) Balb/c to C57BL/6 full-thickness skin transplantation with complete graft rejection (day 18). (D) Syngeneic full-thickness skin transplantation (day 8). (E) Syngeneic full-thickness skin transplantation (day 30). (F) C57BL/10 to C57BL/6 (minor mismatch) full-thickness skin transplantation with chronic graft rejection (day 100).&#160; <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55105/55105fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55105/55105fig3.jpg" /> 
Figure 3: Balb/c to C57BL/6 Skin Graft Survival. (A) Treatment with cyclosporine (25 mg/kg QD). (B) Treatment with rapamycin (3 mg/kg QD). (C) Treatment with metabolic inhibitors, 2-Deoxy-D-glucose (2DG) 500 mg/kg QD, metformin 150 mg/kg QD, and 6-Diazo-5-oxo-L-norleucine (DON) 1.6 mg/kg QOD. All treatments were administered from the day of transplantation until rejection. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55105/55105fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55105/55105fig4.jpg" /> 
Figure 4: Hematoxylin and Eosin Stain (right, X100; left, X200) of Skin Grafts with Metabolic Therapy at Postoperative Day 7. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55105/55105fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Full-thickness Skin Transplantation at JoVE.com

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

murine-full-thickness-skin-transplantation

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

20.0K Views.  Johns Hopkins University School of Medicine.  Murine full-dikte van de huid transplantatie is een gevestigde model om afstoting te studeren in een alloimmuun setting. Hier bieden we een tutorial van elke stap die betrokken zijn bij het uitvoeren van een BALB / c -> C57BL / 6 full-dikte van de huid transplantatie. 

Video: Murine Full-thickness Skin Transplantation

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

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Bioengineering

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As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

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Biology

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Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

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Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medicine

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Developmental Biology

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.

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.

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

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

A Technique for Human Skin Grafting in a Mouse Model

Source: Moss, M. I. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64040">Xenograft Skin Model to Manipulate Human Immune Responses&#160;In Vivo.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates a method for grafting human skin onto an immunodeficient mouse with a suppressed neutrophil response. The method involves applying human split-thickness skin to the mouse&#39;s graft bed, sealing it, and covering it for recovery. Eventually, the human skin adheres to the mouse&#39;s skin, establishing vascularization and circulation, resulting in a successful skin xenograft.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Murine Full-thickness Skin Transplantation

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