Generation of Organotypic Full Skin Reconstructs: A Procedure to Establish 3D Skin Model Using Human Skin Samples

Published: April 30, 2023

Abstract

Source: Müller, I. et al. A 3D Organotypic Melanoma Spheroid Skin Model. J. Vis. Exp. (2018)

This video describes the detailed protocol for generating in vitro 3D organotypic skin reconstructs to study melanoma progression. This skin model mimics both the architecture and multicellular complexity of organs in vivo.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Isolation of Primary Keratinocytes from the Epidermis Wash the epidermal tissue with PBS. Cut the epidermis into smaller pieces (1 mm2) and collect them in a 50-mL conical tube. Add 10 mL 1x Trypsin-EDTA (preheated to 37 °C) an…

Representative Results

Figure 1: Cultivation of 3D organotypic skin reconstructs submerged with medium and at the air-liquid interface. At day 0, primary keratinocytes are seeded on top of the dermal compartment consisting of primary fibroblasts embedded into a collagen type I matrix. 3D skin reconstructs stay cultivated submerged with EGM for 7 days, detach from the insert wall, and start shrinking. At day 8, the inse…

開示

The authors have nothing to disclose.

Materials

fetal serum albumin (FCS) Thermo Fisher Scientific 10270106 add 10 % to cell culture medium
Trypsin-EDTA Thermo Fisher Scientific 25300054 1X dilute in PBS
DMEM Thermo Fisher Scientific 41965062 for cultivation of primary fibroblasts
Dispase Gibco life technologies 17105041 2U/ml, dissolve in PBS
Collagen type I BD Biosciences 354249 usually from rat tail, concentration should be 3.5-4 mg/ml
24-well inserts Nunclon Nunc 140629 8 µm pore size; use standing, not hanging inserts
cell strainer BD Falcon 352360 yellow
Gentamycine Thermo Fisher Scientific 15750060 dissolve in PBS
Keralife keratincyte medium Cell Systems do not add FCS or antibiotics
Collagenase SERVA 17454 NB4 standard grade
juvenile human keratinocytes Cell Systems FC-0007 alternative to own preparation from juvenile foreskin
juvenile human fibroblasts Cell Systems FC-0001 alternative to own preparation from juvenile foreskin
EGF Gibco life technologies 13247-051 add 10 ng/ml only for the generation of EGM medium
Calcium chloride Merck Chemicals 2381 add 1.9 mM for the generation of EGM and 3.8 mM for MM medium
Selenic acid Alfa Aesar 18851 add 53 nM for the generation of EGM and MM medium
Insulin Lilly HI0219 Hum Pen 3ml 100 E.I./ml add 5 µg/ml for the generation of EGM and MM medium
Ethanolamine Sigma-Aldrich E-0135 add 0.1 mM for the generation of EGM and MM medium
P-Ethanolamine Sigma-Aldrich P-0503 add 0.1 mM for the generation of EGM and MM medium
Holo-Transferrine Sigma-Aldrich T-0665 add 5 µg/ml for the generation of EGM and MM medium
Triiodothyronine Sigma-Aldrich T-6397 add 20 pM for the generation of EGM and MM medium
Hydrocortisone Sigma-Aldrich H-088816 add 0.4 µg/ml for the generation of EGM and MM medium
Progesterone Sigma-Aldrich P-8783 add 10 nM only for the generation of EGM medium
Hepes Sigma-Aldrich H-3784 add 15 mM for the generation of EGM and MM medium
Serine Sigma-Aldrich S-4311 add 1 mM for the generation of EGM and MM medium
Choline chloride Sigma-Aldrich C-7017 add 0.64 mM for the generation of EGM and MM medium
dFCS Sigma-Aldrich F-0392 Lot 086K0361 add 2 % for the generation of EGM and MM medium
Adenine Sigma-Aldrich A-9795 add 0.18 mM for the generation of EGM and MM medium
L-Glutamine PromoCell C-42209 add 7.25 mM for the generation of EGM and MM medium
Strontium chloride Sigma-Aldrich 255521 add 1 mM for the generation of EGM and MM medium

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記事を引用
Generation of Organotypic Full Skin Reconstructs: A Procedure to Establish 3D Skin Model Using Human Skin Samples. J. Vis. Exp. (Pending Publication), e20351, doi: (2023).

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