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 |
