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

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…

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