Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

Summary

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.

Abstract

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.

Introduction

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 betwe....

Protocol

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.......

Discussion

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 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.......

Materials

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