Name: Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis (Video) | JoVE Journal
Uploaded: 2015-12-14T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Learn to generate genetically modified organotypic skin cultures using devitalized human dermis and epidermal keratinocytes. This method enables rapid study of gene function in dermatology.

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5 minutes

This work was supportedby the American Cancer Society Research Scholars Grant(RSG-12-148-01-DDC) and the CIRM Basic Biology Award (RB4-05779).

The human skin protocol was performed in accordance with the guidelines of the University of California, San Diego&#8217;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
<ol>
	<li>Upon receiving human skin, place the tissue in the tissue culture hood to thaw (~3-5 min). Depending on the size of the skin, place thawed tissue in a sterile disposable 50 ml conical tube or 125 ml bottle. The typical size of skin from the skin bank is 0.75 square feet.</li>
	<li>Fill the container 75% full with 4x penicillin and streptomycin (pen/strep) in 1x PBS. Shake vigorously for 5 min and dispose of the liquid. Repeat this step 3 more times.</li>
	<li>After the last wash, transfer the tissue to a new tube/bottle and add fresh 4x pen/strep/PBS to fill 75% of the container. Incubate this mixture in a tissue culture incubator at 37 &#176;C for 2 weeks to allow the separation of the dermis from the epidermis.</li>
	<li>Under sterile conditions, use forceps to peel away the epidermis from the dermis. The dermis is completely white while the epidermis will have coloring depending on the race of the donor (Figure 1A). Discard the epidermis according to the institution&#8217;s protocol for dealing with human tissue.</li>
	<li>Place the dermis in a tube or bottle and wash it in 4x pen/strep diluted in 1x PBS with vigorous shaking (5 min washes for 4 times). After the last wash, transfer the dermis to a new tube/bottle in 4x pen/strep diluted in 1x PBS and store at 4 &#176;C until ready to use. Dermis can be stored at 4 &#176;C for up to a year.</li>
</ol>
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.
<ol>
	<li>Day 1: The day before transfection, seed phoenix cells in a 6-well plate at a density of 800,000 cells per well in complete DMEM media.</li>
	<li>Day 2: The day of transfection, use 3 &#181;g of retroviral vectors per well. Aliquot 3 &#181;g of retroviral vector into a 1.5 ml tube. Use a mix of 100 &#181;l DMEM plus 6 &#181;l of transfection reagent for every 3 &#181;g of vector. Mix 6 &#181;l of transfection reagent with 100 &#181;l DMEM in a 1.5 ml tube. (The retroviral vectors that are used are listed in the equipment/materials table).
	<ol>
		<li>Incubate for 5 min and add the 106 &#181;l mixture into the pre-aliquoted tube containing the 3 &#181;g of retroviral vector. Mix and incubate at RT for 30 min. Add this entire mixture dropwise to each well of the phoenix cells.</li>
	</ol>
	</li>
	<li>Day 3: The day after transfection, remove the media from the phoenix cells and add 2 ml of fresh complete DMEM media. On the same day, seed primary human keratinocytes into 6-well plates at a density of 75,000 cells per well. Maintain the keratinocytes in a keratinocyte serum free medium (KCSFM) with antibiotics (pen/strep). 
	NOTE: Primary human keratinocytes were purchased. Cells can be purchased from a variety of vendors (listed in the Material/Equipment table).</li>
	<li>Day 4: Harvest the media from the transfected phoenix cells, which now contains viral particles. Pass the media through a 0.45 &#181;m filter using a syringe (to remove any contaminating phoenix cells) and place 2 ml onto the keratinocytes (plated at 75,000 cells/well the previous day: see step 2.3). Add hexadimethrine bromide (5 &#181;g/ml) to the media to help mediate the infection process. Viral titers are ~1 x 107 TU/ml.
	<ol>
		<li>Spin the 6-well plates in a centrifuge at 200 x g for 1 hr at RT. After the spin, remove the media containing virus and wash the cells once with 1x PBS before adding KCSFM.</li>
	</ol>
	</li>
	<li>Day 5: Infect the same batch of keratinocytes again as in step 2.4. 
	NOTE: Select the keratinocytes using a drug if there is a selectable marker on the retroviral vector and expand for use in downstream analysis or applications such as organotypic cultures.</li>
</ol>
3. Setting Up Organotypic Skin Cultures
<ol>
	<li>Build the organotypic cassette from common materials found in the laboratory or the cassettes can be custom made through a metal fabrication shop (Figure 2A-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 2A-B). Do this under sterile conditions.
	<ol>
		<li>Attach square pegs to the bottom of the cassette (lid of 3.5 cm dish) using clear nail polish (Figure 2C). Allow 5 min for the nail polish to dry and flip the cassette over so that it is resting on the square pegs (Figure 2D). Place the cassette into a 6 cm dish. 
		NOTE: Square pegs can be purchased from a variety of arts and crafts stores.</li>
	</ol>
	</li>
	<li>Prepare keratinocyte growth medium (KGM) comprised of 66% DMEM, 22% Ham&#8217;s F12, 100 units/ml penicillin, 100 &#181;g/ml streptomycin, 10% FBS, 2.67 &#181;g/ml adenine, 40 ng/ml hydrocortisone, 10 ng/ml cholera toxin, 4.94 &#181;g/ml insulin, 5 &#181;g/ml transferrin, 1.36 ng/ml Triiodo-L-thyronine, 10 ng/ml EGF, and 10 &#181;g/ml ciprofloxacin hydrochloride. Filter the media through a 0.22 &#181;m filter and store at 4 &#176;C until ready to use.</li>
	<li>Take the dermis out of the 4x pen/strep + PBS solution and wash 2 times in KGM and then incubate in KGM at 37&#160;&#176;C for two days prior to use.</li>
	<li>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 the keratinocytes (Figure 1B).</li>
	<li>Place the pre-cut dermis onto the square hole of the organotypic cassette with the top side of the dermis facing up (Figure 3A).</li>
	<li>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 &#181;l 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).</li>
	<li>Allow 5 min for the commercial extra cellular 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.</li>
	<li>Place 0.5-1 million genetically modified keratinocytes onto the organotypic cassettes containing the dermis (Figure 3D).
	<ol>
		<li>Trypsinize 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.</li>
		<li>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 &#181;l 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 differences seen in thickness of the regenerated epidermis is not due to differences in starting cell number. Placing less than 0.5 million cells on dermis can lead to poor stratification and differentiation.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>

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</ol>

We have nothing to disclose.

Genetic manipulation in human skin organotypic cultures offer many advantages to commonly studied 2D cultured cells as well as mouse models. 2D cultures lack the three dimensional cell-cell and cell-extracellular matrix interactions found in intact tissues and organs. Recent studies have also found tremendous differences between 2D and 3D cultured skin cancer cells with the 3D cultured cells showing much more gene expression similarities to primary human skin tumors16. Human organotypic skin cultures also have...

The human epidermis is a stratified epithelium that connects to the underlying dermis through an extracellular matrix known as the basement membrane zone.The epidermis not only serves as an impermeable barrier to prevent the loss of moisture but also as a first line of defense to protect the body from foreign and toxic substances1. The basal layer, which is the deepest layer of the epidermis, contains the epidermal stem and progenitor cells that give rise to the differentiated progeny that form the rest of the epidermis2. As epidermal progenitor cells differentiate they migrate upwards to form the first layer of differentiated cells known as the spinous layer3. In the spinous layer, cells turn on the expression of keratins 1 and 10, which then provide the strength to withstand physical stress for the differentiated layers of the epidermis. As the spinous layer cells further differentiate, they move upwards in the epidermis to form the granular layer which is characterized by the formation of keratohyalin and lamellar granules as well as structural proteins that are assembled below the plasma membrane. As the cells proceed in the differentiation process, the proteins beneath the plasma membrane are cross linked to each other while the lamellar granules are extruded from the cells to form a lipid rich barrier called the stratum corneum4.
Diseases that involve alterations in epidermal growth and differentiation impact ~20% of the population5. Thus, understanding the mechanisms of this process is of great importance. Since manifestation of many of these diseases is contingent upon cell-cell or cell-matrix contact, organotypic cultures where the human epidermis is reconstituted in a 3D environment have been created6-10. These methods typically involve the use of primary or transformed keratinocytes seeded on extracellular matrix such as devitalized human dermis, Matrigel, or collagen.
To understand gene regulatory mechanisms that are important in epidermal growth and differentiation, keratinocytes can be genetically manipulated through retroviral vectors to knockdown or overexpress genes in 2D culture and then reconstituted in 3D. These methods have been used extensively to characterize genes involved in epidermal stem and progenitor cell self-renewal and differentiation as well as progression to neoplasia11-21. Here, an in-depth protocol on how to alter gene expression in epidermal organotypic cultures through the use of retroviruses is provided.

<table><tbody><tr><td>Human skin</td><td>New York Firefighters Skin Bank</td><td>&lt;a href=&quot;http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html&quot;&gt;http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html&lt;/a&gt;</td><td></td></tr><tr><td>PEN/STREP</td><td>GIBCO</td><td>15140-122</td><td></td></tr><tr><td>amphotropic phoenix cell lines</td><td>ATCC</td><td>CRL-3213</td><td></td></tr><tr><td>FUGENE 6 transfection reagent</td><td>Promega</td><td>E2691</td><td></td></tr><tr><td>Keratinocyte Media (KCSFM)</td><td>Life Technologies</td><td>17005042</td><td></td></tr><tr><td>DMEM</td><td>GIBCO</td><td>11995</td><td></td></tr><tr><td>Ham&#039;s F12</td><td>Cambrex</td><td>12-615F</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>10437-028</td><td></td></tr><tr><td>Adenine</td><td>Sigma</td><td>A-9795</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma</td><td>&amp;nbsp;C-8052</td><td></td></tr><tr><td>Hydrocortisone</td><td>Calbiochem</td><td>3896</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I-1882</td><td></td></tr><tr><td>EGF</td><td>Invitrogen</td><td>13247-051</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma</td><td>T-0665</td><td></td></tr><tr><td>Ciprofloxacin Hydrochloride</td><td>Serologicals</td><td>89-001-1</td><td></td></tr><tr><td>cautery</td><td>Bovie Medical Corporation</td><td>AA01</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354234</td><td></td></tr><tr><td>Keratin 1 antibody</td><td>Biolegend</td><td>PRB-149P</td><td></td></tr><tr><td>square pegs</td><td>Arts and crafts stores</td><td></td><td></td></tr><tr><td>human neonatal keratinocytes</td><td>ATCC</td><td>PCS-200-010</td><td></td></tr><tr><td>human neonatal keratinocytes</td><td>Cell Applications</td><td>102K-05n</td><td></td></tr><tr><td>MSCV retroviral vector</td><td>Clontech</td><td>634401</td><td></td></tr><tr><td>LZRS retroviral vector</td><td>Addgene</td><td></td><td></td></tr><tr><td>pSuper.Retro.Puro Retroviral vector</td><td>Oligoengine</td><td>VEC-PRT-0002&amp;nbsp;</td><td></td></tr><tr><td>hexadimethrine bromide&amp;nbsp;</td><td>Sigma</td><td>H9268-5G</td><td></td></tr></tbody></table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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.

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 t...

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

The overall goal of this procedure is to generate genetically modified organotypic skin cultures using revitalized human dermis to determine the impact of altered gene expression on epidermal growth and differentiation. This method can help answer key questions in the dermatology field, such as determining which genes are responsible for epi growth, differentiation, and progression to disease. The main advantage of this technique is that genetically altered human skin is generated within a few days, which is allows the study of human samples that are potentially clinically relevant.Demonstrating the procedure will be jingly from my laboratory. If receiving frozen human skin, place the tissue in the tissue culture hood for three to five minutes to thaw. Once thawed, transfer the tissue into a sterile 50 milliliter conical tube or 125 milliliter bottle, depending on size.Next, add four x penicillin and streptomycin in one XPBS until the container is 75%full. Cap the container and shake vigorously for five minutes. Remove the antibiotic solution and repeat this.Step three more times after the last wash, transfer the tissue to a new container and add fresh antibiotic solution. Incubate this mixture in a tissue culture incubator at 37 degrees Celsius for two weeks to allow the separation of the dermis from the epidermis. When two weeks have elapsed, use forceps to peel away the epidermis from the dermis.Discard the epidermis according to the institution's protocol for dealing with human tissue. Place the dermis in a container of antibiotic solution and wash with vigorous shaking as before. After the last wash, transfer the dermis to a new container of antibiotic solution and store at four degrees Celsius until ready to use the day before transfection seed phoenix cells in complete media into a six well plate at a density of 800, 000 cells per well.On the day of transfection, mix six microliters of transfection reagent with 100 microliters of dmm. For each well to be transfected and incubate at room temperature For five minutes, add the mixture to a tube containing three micrograms of retroviral vector for each well to be transfected and incubate at room temperature for 30 minutes, add this entire mixture dropwise to each well of the phoenix cells and incubate the cells overnight the day after transfection. Remove the media from the phoenix cells and add two milliliters of fresh complete media on the same day.Resuspend primary human keratinocytes in keratinocyte serum free medium with antibiotics and seed into six wall plates at a density of 75, 000 cells per well. The following day, harvest the media which now contains viral particles from the transfected Phoenix cells. Pass the media through a 0.45 micron filter using a syringe to remove any contaminating cells.Add five micrograms per milliliter of hexa dimethyl bromide to the filtered virus containing media to help mediate the infection process. Then pipette two milliliters of the virus onto the keratinocytes plated the previous day. Next, spin the six wall plates in a centrifuge at 200 Gs for one hour at room temperature.After the spin, remove and discard the media containing virus. Wash the cells once with one XPBS and then add fresh K-C-S-F-M the following day. Infect the same batch of keratinocytes with virus using the procedure just shown.To make organotypic cassettes. Use a cautery to remove a one centimeter by one centimeter square from the center of the lid of a 3.5 centimeter tissue culture dish. Attach square pegs to the bottom of the cassette using clear nail polish and allow five minutes for the nail polish to dry.Finally, flip the cassette over and place the cassette into a six centimeter dish with a cassette resting on the pegs. Retrieve the dermis from four degrees Celsius storage and transfer it to a container of keratinocyte growth. Medium to wash.After repeating the wash, place the dermis in fresh KGM and incubate at 37 degrees Celsius for two days. Following the incubation, use a scalpel to cut the dermis into 1.5 centimeter by 1.5 centimeter sized pieces. Then place onto the square hole of the organotypic is set with the top of the dermis facing up.Use forceps to flip the entire organotypic cassette containing the dermis. Add five drops of freshly ough extracellular matrix to the bottom of the dermis and shake slightly to ensure even distribution across the dermis. After allowing the extracellular matrix to solidify for five minutes, use forceps to flip the cassette back over, and then add four milliliters of KGM to the six centimeter dish.Next, after harvesting and counting the genetically modified keratinocytes Resus suspend 0.5 to 1 million cells in 90 microliters of KGM and dispense the cells onto the dermis. Incubate the co-culture at 37 degrees Celsius. Change the media on the cultures every other day.Full stratification and differentiation of the epidermis usually occurs after day five. Tissue can be harvested up to 14 days after plating. This image shows hematin and eosin staining of organotypic.Skin cultures regenerated human skin contains both the epidermis and dermis. The epidermis is fully stratified and differentiated with four distinct layers, including the undifferentiated basal layer and the three differentiated layers including the stratum, spinosum, ranum, and cornium. These immunofluorescence images demonstrate that overexpression of SNA i two as shown on the right inhibits epidermal differentiation compared to control tissue shown on the left staining for the differentiation.Protein keratin one or K one is shown in green and nuclei is marked in blue. RTQ PCR R analysis to measure mRNA levels of SNAI two in control LAC Z and S NNA I two over expressing tissue demonstrates greater than 50 fold expression of SNAI two relative to control. Finally, this RTQ PCR R analysis demonstrates that overexpression of SNA I two results in loss of expression of differentiation induced structural genes such as TGM one and SPRR one A.Following this procedure.Other methods like chipsy, immunofluorescence, RN, ae, or western blotting can be performed in order to determine the mechanisms of epidural growth and differentiation. This technique paved the way for researchers in the field of dermatology to explore how alter gene function impacts skin homeostasis. After watching this video, you should have a good understanding of how to generate genetically modified organic human skin cultures using keratinocytes and dev human dermis.

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Learn to generate genetically modified organotypic skin cultures using devitalized human dermis and epidermal keratinocytes. This method enables rapid study of gene function in dermatology.

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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