Organotypic 3D Skin Culture: A Cassette-based In Vitro Culture Technique to Regenerate Human Epidermis on Acellular Dermis

Overview

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.

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. Setting Up Organotypic Skin Cultures

1. Build the organotypic cassette from common materials found in the laboratory or the cassettes can be custom-made through a metal fabrication shop (Figure 1A-F). To make these cassettes from a 3.5 cm tissue culture plate, 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 (Figure 1A-B). Do this under sterile conditions.
   1. Attach square pegs to the bottom of the cassette (lid of 3.5 cm dish) using clear nail polish (Figure 1C). Allow 5 min for the nail polish to dry and flip the cassette over so that it is resting on the square pegs (Figure 1D). Place the cassette into a 6 cm dish.
      NOTE: Square pegs can be purchased from a variety of arts and crafts stores.
2. Prepare a keratinocyte growth medium (KGM) comprising of 66% DMEM, 22% Ham’s F12, 100 units/mL penicillin, 100 µg/mL streptomycin, 10% FBS, 2.67 µg/mL adenine, 40 ng/mL hydrocortisone, 10 ng/mL cholera toxin, 4.94 µg/mL insulin, 5 µg/mL transferrin, 1.36 ng/mL Triiodo-L-thyronine, 10 ng/mL EGF, and 10 µg/mL ciprofloxacin hydrochloride. Filter the media through a 0.22 µm filter and store at 4 °C until ready to use.
3. Take the dermis out of the 4x pen/strep + PBS solution and wash 2 times in KGM and then incubate in KGM at 37 °C for two days prior to use.
4. Cut the dermis using a scalpel to 1.5 cm x 1.5 cm sized pieces and then place onto the organotypic cassette. Place the dermis with the top of the dermis facing up. The top of the dermis will be the side that comes into contact with keratinocytes (Figure 2A).
5. Place the pre-cut dermis onto the square hole of the organotypic cassette with the top side of the dermis facing up (Figure 3A).
6. Flip the entire organotypic cassette containing the dermis over using forceps (Figure 3B). Thaw the extracellular matrix solution on ice. Use a needle and syringe to draw out 100 µL of extracellular matrix solution per cassette. Add 5 drops to the bottom of the dermis (glossy side) and shake slightly to ensure even distribution throughout the dermis (Figure 3B).
7. Allow 5 min for the commercial extracellular matrix solution to completely solidify (Figure 3C). After solidification, use forceps to flip the cassette back over. Add 4 mL of KGM to the 6 cm dish.
8. Place 0.5-1 million genetically modified keratinocytes onto the organotypic cassettes containing the dermis (Figure 3D).
   1. Trypsinize the keratinocytes by placing 4 mL of 0.05% Trypsin onto 10 cm plates for 5 min and quench with 4 mL of complete DMEM media.
   2. Count the keratinocytes using a hemocytometer and then spin down at 200 x g for 5 min. Resuspend 0.5-1 million cells (depending on the number of cells available) in 90 µL of KGM and place all the cells onto the dermis.
      NOTE: It is important to place the same number of cells on dermis within the same experiment to ensure that any difference seen in the thickness of the regenerated epidermis is not due to the differences in starting cell number. Placing less than 0.5 million cells on the dermis can lead to poor stratification and differentiation.
9. Change KGM media on the organotypic cultures every other day. Harvest tissue 1-14 days after placement of keratinocytes on the dermis. Full stratification and differentiation of the epidermis usually occurs after day 5.

Representative Results

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.

Materials

Name	Company	Catalog Number	Comments
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