人体皮肤外植体制备和培养

Jessica L. Shannon; Stephen J. Kirchner; Jennifer Y. Zhang

doi:10.21769/BioProtoc.4514

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Peer-reviewed

Human Skin Explant Preparation and Culture

人体皮肤外植体制备和培养

JS Jessica L. Shannon

SK Stephen J. Kirchner

JZ Jennifer Y. Zhang email

发布: 2022年09月20日第12卷第18期 DOI: 10.21769/BioProtoc.4514 浏览次数: 2727

评审: Giusy TornilloDongsheng JiangGiovanna Piovani

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Abstract

The ex vivo experimentation with surgically discarded human skin represents a unique methodology amenable for mechanism and pharmacologic agent studies without the involvement of human subjects. Here, we describe a protocol that includes preparation, culture, and stimulation of human skin explants, and subsequent analyses by quantitative reverse transcription PCR and immunostaining. This protocol may also be applied for ex vivo studies of murine skin, reducing animal numbers and potentially harmful treatments. In our hands, this protocol has been used for wound healing, viral infection, and hair growth–related studies.

Graphical abstract:

Cartoon of explant skin culture. Skin explant sits on top of a gelatin surgical sponge saturated with culture medium at an air–liquid interface.

Keywords: Human skin (人体皮肤)

Tissue explant (组织外植体)

Organ culture (器官培养)

Ex vivo (离体)

Cutaneous immunity (皮肤免疫)

Cutaneous wound healing (皮肤伤口愈合剂)

Background

Genetic animal models are powerful in recapitulating complex tissue environment with intact circulation. Mice are by far the most frequently used animal for gene function, disease modeling, and pharmacological studies. However, murine skin and human skin are architecturally different and show differences in their responses to injury (Zomer and Trentin, 2018). It is not surprising that findings using murine skin often need corroboration in human skin models that harbor tissue-resident immune cells (Zomer and Trentin, 2018).

Significant technical advances have been achieved in epidermal keratinocyte culture in vitro, allowing procurement and expansion of primary keratinocytes. However, monolayer cell culture does not always recapitulate in vivo processes and often lacks tissue-resident immune cells. The organotypic cell culture model generated with primary or immortalized keratinocytes allows three-dimensional epidermal stratification and incorporation of certain dermal cells such as fibroblasts (Smits et al., 2017). Each of these methods has limitations in recapitulating skin biology.

Our lab has adapted a protocol for using surgically discarded human skin samples for ex vivo studies (Shannon et al., 2022), such as wound assays, cytokine stimulation, and viral infections. The protocol described below includes necessary materials, reagents, equipment, and procedures.

Materials and Reagents

1.7 mL microtube (Genesee Scientific, catalog number: 22-281LR)
SURGIFOAM^® absorbable gelatin sponge (Ethicon, catalog number: 1969)
12-well cell culture plates, flat bottom wells, TC-treated (GenClone 25-106)
Sterile 1× phosphate buffered saline (PBS) (Gibco, catalog number: 14190144)
Penicillin–streptomycin–glutamine (100×) (Gibco, catalog number: 10378016)
EpiLife^TM CF kit (Gibco, catalog number: MEPICF500)
Note: This kit contains 500 mL of EpiLife^TM calcium-free media and 500 μL of 0.06 M CaCl₂(see Recipes).
Human keratinocyte growth supplement (HKGS, 100×) (Gibco, catalog number: S0015)
Adwin scientific Tissue-Tek Cryomold, intermediate (Fisher Scientific, catalog number: NC9511236)
Tissue-Tek optimum cutting temperature (OCT) compound (VWR, catalog number: 25608-930)
Rat anti-human Integrin α6 CD49f antibody (BioLegend, catalog number: 313602, diluted 1:500)
Alexa Fluor 647-conjugated goat anti-rat secondary antibody (BioLegend, catalog number: 313609). Diluted 1:400
Prolong Gold antifade reagent (ThermoFisher Scientific, catalog number: P36930)
TRIzol^TM reagent (ThermoFisher Scientific, catalog number: 15596026)
Hoechst 33342, trihydrochloride, trihydrate (ThermoFisher Scientific, catalog number: H3570)
iScrip^TM cDNA synthesis kit (Bio-Rad, catalog number: 1708891)
Culture medium (see Recipes)
Wash buffer (see Recipes)
70% ethanol (see Recipes)

Equipment

CO₂ incubator (Panasonic, model: MCO-170AICUVL-PA)
Technocut^® disposable scalpels, sterile, MYCO Medical, No. 20 (VWR, catalog number: 10148-894)
Forceps (VWR, catalog number: 89259-946)
Surgical scissors (VWR, catalog number: 76192-134)
Pipettes (ThermoFisher Scientific, model: Finnpipette^TM F2 variable volume pipettes, catalog number: 4701070)
Olympus IX73 fluorescent microscope (Olympus Corporation Microscopy Technologies)
Nanodrop 1000
-80 °C freezer
Leica Cryocut 1800

Procedure

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如何引用

Shannon, J. L., Kirchner, S. J. and Zhang, J. Y. (2022). Human Skin Explant Preparation and Culture. Bio-protocol 12(18): e4514. DOI: 10.21769/BioProtoc.4514.