Source: Jingting Li et al. Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis, J. Vis. Exp. (2015).
This video protocol describes a cassette-based in vitro culture technique to generate human organotypic skin using epidermal keratinocytes and devitalized human dermis. They mimic the function and physiology of their in vivo tissue counterparts.
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. Setting Up Organotypic Skin Cultures
Figure 1. Generation of organotypic cassettes. (A) Use a cautery to remove a 1.0 cm x 1.0 cm square from the center of the lid of a 3.5 cm dish. (B) Remove the square from the center of the 3.5 cm dish. (C) Use clear nail polish to adhere square pegs. Allow 5 min for the nail polish to dry. (D) Flip the lid over so that it is resting on the square pegs. The cassette is now ready to be used. (E) Image of a metal fabricated organotypic cassette with its dimensions. (F) Image of a metal fabricated cassette flipped upside down to show the support pegs.
Figure 2. Preparation of the dermis from human skin. (A) After incubation of the human skin at 37 °C for 2 weeks, the dermis can be separated from the epidermis. Forceps are used to peel the epidermis (brown colored) away from the dermis (white). (B) The top of the dermis can be distinguished from the bottom by the glossiness of the tissue. The top of the dermis (the side that will naturally face the epidermis in vivo or where the keratinocytes will be seeded) is non-glossy. The bottom of the dermis is glossy. The tissue is pink due to incubation in KGM.
Figure 3: Generation of organotypic skin cultures. (A) Place the dermis with the top of the dermis (non-glossy side) facing up onto the organotypic cassette. (B) Flip the cassette over and add Matrigel to the glossy side of the dermis. (C) Allow 5 min for the Matrigel to solidify and then flip the cassette back over. (D) Add genetically modified keratinocytes to the dermis (0.5-1 million cells are resuspended in 90 µl of KGM). Harvest the tissue 1-14 days after seeding of the keratinocytes.
The authors have nothing to disclose.
Human skin | New York Firefighters Skin Bank | http://www.cornellsurgery.org/ pro/services/burn-surgery/skinbank.html | |
Fetal calf serum | HyClone | SH30071.03 | |
MEM Non-Essential Amino Acids Solution, 100X | Thermo Fisher Scientific | 11140050 | |
GLUTAMAX I, 100X | Thermo Fisher Scientific | 35050061 | L-Glutamine |
DPBS, no calcium, no magnesium | Thermo Fisher Scientific | 14190136 | |
0.05% Trypsin-EDTA | Thermo Fisher Scientific | 25300-054 | |
DMEM/F12 medium | Thermo Fisher Scientific | 11330-032 | |
DMEM | GIBCO | 11995 | |
Ham's F12 | Cambrex | 12-615F | |
FBS | GIBCO | 10437-028 | |
Adenine | Sigma | A-9795 | |
Cholera Toxin | Sigma | C-8052 | |
Hydrocortisone | Calbiochem | 3896 | |
Insulin | Sigma | I-1882 | |
EGF | Invitrogen | 13247-051 | |
Transferrin | Sigma | T-0665 | |
Ciprofloxacin Hydrochloride | Serologicals | 89-001-1 | |
cautery | Bovie Medical Corporation | AA01 | |
Matrigel | Corning | 354234 | |
Keratin 1 antibody | Biolegend | PRB-149P | |
square pegs | Arts and crafts stores | ||
PEN/STREP | GIBCO | 15140-122 | |
Human neonatal keratinocytes | ATCC | PCS-200-010 | |
Human neonatal keratinocytes | Cell Applications | 102K-05n | |
MSCV retroviral vector | Clontech | 634401 | |
Keratinocyte Media (KCSFM) | Life Technologies | 17005042 |