The human skin protocol was performed in accordance with the guidelines of the University of California, San Diego’s Research Ethics Committee. Human skin can be obtained from discarded surgical samples or bought from skin banks (skin bank is listed in the Material/Equipment Table). The location where the skin is derived from or age of the donor is not critical to the experiment as long as the basement membrane zone proteins (collagen/laminin) in the dermis are not degraded.
1. Preparation of Devitalized Human Dermis
2. Genetically Modifying Primary Human Keratinocytes
NOTE: Use amphotropic phoenix cells grown in complete DMEM media [DMEM +10% fetal bovine serum (FBS)+pen/strep] to produce viruses to infect primary human keratinocytes. Viral packaging genes that encode proteins such as gag-pol and envelope are stably integrated into the phoenix cell genome which allows for virus production when transfected with a retroviral vector. Phoenix cells can be transfected with high efficiency allowing for high viral titer production. Virus produced from this packaging line can also infect a large variety of mammalian cells including human.
3. Setting Up Organotypic Skin Cultures
The first step in generating organotypic human skin is to remove the epidermis from the dermis. The two week incubation of the skin at 37 °C in 4x pen/strep/PBS should allow the separation of the dermis from the epidermis (Figure 1A). If separating the epidermis and dermis is difficult then place the tissue at 37 °C in 4x pen/strep/PBS for another week and then try peeling again using forceps.
One of the keys to regenerating human epidermis on devitalized dermis is to ensure that the keratinocytes are placed on the correct side of the dermis. The dermis has a top and a bottom. The top of the dermis is the side that comes into contact with the keratinocytes in vivo as well as in organotypic cultures. The top of the dermis can be differentiated from the bottom of the dermis due to the glossiness of the bottom (Figure 1B). If keratinocytes are placed directly onto the bottom side (glossy) of the dermis, the epidermis will not differentiate or stratify properly. Thus, it is crucial to have the top of the dermis (non-glossy) facing up in the organotypic cassette. The dermis can be placed directly onto an organotypic cassette (Figure 2A–F). Cassettes made from tissue culture plates should be UV sterilized while metal fabricated cassettes can be autoclaved prior to the dermis being placed on. The genetically modified keratinocytes can then be placed onto the dermis. The keratinocytes are initially submerged in liquid since they were placed onto the dermis resuspended in KGM. After 24 hr the KGM will have diffused through the dermis which allows the keratinocytes to be exposed to the air-liquid interface which causes stratification and differentiation into epidermis7 (Figure 3).
RNA, protein, DNA/chromatin, as well as tissue sections can be harvested from the organotypic skin cultures which can all be used for a variety of downstream applications. RNA can be used to determine gene expression differences between control and knockdown/overexpression tissue using RT-QPCR, microarrays, or RNA-Seq. DNA/chromatin can be used for DNA or ChIP-Seq applications. The regenerated skin can be mounted in OCT and frozen sections can be used for immuno-staining or hematoxylin and eosin staining to assess tissue morphology, protein expression/localization in control and experimental groups.
An example of this is shown in Figure 4 with the hematoxylin and eosin staining of a typical organotypic skin culture. Note that all four distinct layers of the epidermis are formed with layer specific expression of differentiation related proteins (Figure 4)11,12,14,15. This system can be used to determine the impacts of overexpression or knockdown of different genes on the epidermis. Figure 5 shows regenerated epidermal tissue that expresses control, LACZ as well as tissue overexpressed for SNAI2. Overexpression of SNAI2 led to an inhibition of epidermal differentiation as noted by the lack of keratin 1 (K1) staining as well as loss of expression of differentiation induced structural genes such as TGM1 and SPRR1A (Figure 5A–C)13,22. The epidermal thickness shown in Figure 4 and Figure 5 may vary within the same sample due to more cells accumulating in the middle of the dermis versus the periphery when the cells were initially placed on the dermis. Thus, the middle sections tend to have thicker epidermis whereas the periphery tends to have thinner. For direct comparison between different samples the same regions of a section should be compared. However, the staining for the markers should stay consistent.
Figure 1. 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. Please click here to view a larger version of this figure.
Figure 2. 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 4. Hematoxylin and eosin staining of organotypic skin cultures. Regenerated human skin contains both the epidermis and dermis. The epidermis is fully stratified and differentiated with 4 distinct layers. These layers include the undifferentiated basal layer and the 3 differentiated layers including stratum spinosum, granulosum and corneum. Please click here to view a larger version of this figure.
Figure 5. Immunofluorescence staining and gene expression analysis of genetically modified human epidermis. (A) Overexpression of SNAI2 inhibits epidermal differentiation. Staining for differentiation protein keratin 1 (K1) is shown in green and nuclei is marked in blue (Hoechst). (B) RT-QPCR analysis on the mRNA levels of SNAI2 in control LACZ and SNAI2 overexpressing tissue. Error bars = SD, n = 3. (C) RT-QPCR analysis on the mRNA levels of differentiation transcripts KRT1, TGM1, and SPRR1A in control LACZ and SNAI2 overexpressing tissue. Error bars = SD, n = 3. Please click here to view a larger version of this figure.
Human skin | New York Firefighters Skin Bank | http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html |
PEN/STREP | GIBCO | 15140-122 |
amphotropic phoenix cell lines | ATCC | CRL-3213 |
FUGENE 6 transfection reagent | Promega | E2691 |
Keratinocyte Media (KCSFM) | Life Technologies | 17005042 |
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 | |
human neonatal keratinocytes | ATCC | PCS-200-010 |
human neonatal keratinocytes | Cell Applications | 102K-05n |
MSCV retroviral vector | Clontech | 634401 |
LZRS retroviral vector | Addgene | |
pSuper.Retro.Puro Retroviral vector | Oligoengine | VEC-PRT-0002 |
hexadimethrine bromide | Sigma | H9268-5G |
Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.
Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.
Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.