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

  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
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.

Divulgaciones

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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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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