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

Overview

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

1. 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) and incubate for 5 min in a water bath at 37 °C. Vortex the tissue suspension every 2 min.
2. Stop the enzymatic reaction by adding 1 mL fetal calf serum (FCS). Resuspend the cells by pipetting up and down with a 10-mL plastic pipette for 4 min. Try to avoid bubbles and foam formation.
3. Pipet the cell suspension into a cell strainer (pore size 100 µm), placed on top of a fresh 50 mL conical tube, and rinse 3x with 5 mL PBS. Centrifuge the cells at 200 x g for 5 min. Resuspend the cell pellet in 2-6 mL culture medium containing 1% (v/v) gentamycin. Determine the cell number manually by counting cells in a hemocytometer and seed 6 x 10⁵ cells into a T75 cell culture flask.

2. Isolation of Primary Fibroblasts from the Dermis

1. Cut the dermal tissue into smaller pieces (1 mm²) and transfer them into a 50-mL conical tube. Add 10 mL collagenase-solution (5 U/mL in PBS⁺) and incubate for 45 min in a water bath at 37 °C. Centrifuge the mixture of tissue pieces and cells at 200 x g for 5 min.
2. Wash the cell pellet twice with 10 mL DMEM containing 4.5 g/L glucose and L-Glutamine, but without L-Pyruvate. Finally, resuspend the tissue pieces in 2 mL of DMEM containing 1% (v/v) gentamycin and 10% FCS. Transfer them into a T25 cell culture flask and incubate in a cell incubator at 37 °C and 5% CO₂ overnight.
   NOTE: The small volume allows the fibroblasts to migrate out of the tissue debris and forces them to attach to the culture flask.
3. The next morning, add another 6 mL DMEM/Gentamycin/FCS and incubate for 2-3 days at 37 °C until the cells have reached 80-90% confluency.

3. Generation of the Dermal Compartment of Organotypic Full Skin Reconstructs

1. Generate skin models using 24-well cell microporous membrane inserts (pore size 8 µm) placed in 24-well plates.
   NOTE: Make sure not to use hanging but standing inserts, because they will be placed into a 6-well plate for air-liquid cultivation at a later stage.
2. Prepare the gel neutralization solution (GNL), as well as the culture media referred to as MM and endothelial growth media (EGM). To prevent coagulation, store the collagen type I (usually from rat tail, 3.5-4 mg/mL in 0.02 N acetic acid) on ice until use because it starts to gel at RT.
3. Re-suspend 1 x 10⁵ fibroblasts per insert in GNL and quickly mix the cell suspension with collagen at a ratio of 1:3 in a final volume of 500 µL/insert. Mix by gently pipetting up and down to avoid bubble formation as bubbles may impair the quality of the skin reconstruct.
4. Allow the individual dermal gels to settle by keeping them without medium at RT for 30 min in a sterile hood. Subsequently, cover each gel with DMEM containing 4.5 g/L glucose, 1% L-glutamine, 10% FCS, and without L-pyruvate, and incubate overnight at 37 °C.

4. Generation of the Epidermal Compartment of Organotypic Full Skin Reconstructs

1. The next day, remove the medium from the dermal gels and equilibrate them with EGM media (10% FCS, 1% PenStrep, 10 mg/mL gentamicin) for 2 h at 37 °C. Withdraw the medium and carefully seed 1 x 10⁵ keratinocytes resuspended in 100 µL EGM on top of the dermal gel.
2. Incubate the reconstructs for 1.5 h at 37 °C to allow the keratinocytes to adhere to the dermal compartment.
   NOTE: During this incubation time, the gels will start to shrink due to fibroblast-induced contraction, and thus, at least partially detach from the insert walls.
3. Cover the skin equivalents with approximately 800 µL EGM and carefully remove the residual gel from the insert wall with a small (white) pipette tip. Culture the skin equivalents submerged in EGM for 7 days in a cell incubator at 37 °C, 5% CO₂, and change the medium every other day.
   NOTE: During this time, the skin equivalents will shrink significantly.

5. Air-liquid Cultivation of Organotypic Full Skin Reconstructs

1. At day 8, transfer each insert into an individual well of a 6-well plate. Only add 1.2-1.4 mL of MM medium to each well, so that the skin reconstruct is supplied with medium from the bottom of the well but is not covered with the medium.
   NOTE: Cultivation at the air-liquid interface allows stratification of the epidermal part and establishment of a full cornified layer (stratum corneum).
2. During the next 10-17 days, change the medium as stated in section 5.1 every other day. During this period, add drugs or other stimuli as needed to the medium. Carefully remove the full skin reconstruct from the microporous membrane insert using curved tweezers for further analysis, e.g., immunohistochemical analysis (Figure 1).

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 inserts are transferred to 6-wells and cultivated at the air-liquid interface to allow epidermal stratification.

Materials

Name	Company	Catalog Number	Comments
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