Name: a 3d organotypic melanoma spheroid skin model
Uploaded: 2018-05-18T13:00:00.000+00:00
Duration: 8 min 49 s
Description: Watch this Scientific Journal Video about A 3D Organotypic Melanoma Spheroid Skin Model at JoVE.com

The authors thank Hanna Voersmann for establishing the 3D organotypic melanoma-spheroid-skin-model and providing excellent protocols. The authors also thank Silke Busch for valuable technical support. The work was supported by BMBF e:Med program "Melanoma Sensitivity" 031A423A.

All human tissue work was performed using approved institutional protocols.
1. Separation of Dermis and Epidermis from Human Skin (Juvenile Foreskin from Circumcision)
<ol>
	<li>Place the foreskin (usually 1 - 2 cm2) into a non-adhesive sterile cell culture dish (&#216; 10 cm) and cover it with 10 - 12 mL of phosphate-buffered saline+ (PBS, 0.5 mM MgCl2, 0.9 mM CaCl2, pH 7.2 = PBS+).Remove all excess (adipose) tissue using sterile scalpel and forceps.</li>
	<li>Cut the skin into 3 mm x 5 mm pieces, wash them with PBS, and transfer them to a non-adhesive sterile cell culture dish (&#216; 6 cm). Cover the tissue pieces with 5 - 7 mL of dispase solution (2 U/mL in PBS). Seal the cell culture dish with paraffin film and incubate for 16 h at 4 &#176;C.</li>
	<li>Retract the epidermal from the dermal part with two sterile forceps. Transfer the dissected tissue pieces to individual non-adhesive cell culture dishes (&#216; 6 cm) and cover each of them with PBS+.</li>
</ol>
2. Isolation of Primary Keratinocytes from the Epidermis
<ol>
	<li>Carefully remove the PBS+ from the cell culture dish using a Pasteur pipette and wash the epidermal tissue pieces (section 1.3) 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 &#176;C) and incubate for 5 min in a water bath at 37 &#176;C. Vortex the tissue suspension every 2 min.</li>
	<li>Stop the enzymatic reaction by adding 1 mL fetal calf serum (FCS). Resuspend the cells by pipetting up and down with a 10-mL plastic pipette for 4 min. Try to avoid bubbles and foam formation.</li>
	<li>Pipet the cell suspension into a cell strainer (pore size 100 &#181;m), placed on top of a fresh 50 mL conical tube, and rinse 3x with 5 mL PBS. Centrifuge the cells at 200 x g for 5 min. Resuspend the cell pellet in 2 - 6 mL culture medium containing 1% (v/v) gentamycin. Determine the cell number manually by counting cells in a hemocytometer and seed 6 x 105 cells into a T75 cell culture flask.</li>
</ol>
3. Cultivation of Primary Keratinocytes
<ol>
	<li>Incubate primary keratinocytes isolated in step 2.3 in a cell incubator at 37 &#176;C and 5% CO2 in keratinocyte medium without FCS, to 50 - 70% confluency.</li>
	<li>To passage the keratinocytes carefully remove the medium, add 5 mL PBS-EDTA, and incubate the cells for 10 min at 37 &#176;C. Check if cells have started to detach from the culture flask under a light microscope (4X&#160;magnification: cells will appear rounded but are still attached!). If not, replace old PBS-EDTA with fresh PBS-EDTA and re-incubate for another 10 min at 37 &#176;C.</li>
	<li>Add 5 mL 1x Trypsin-EDTA to the rounded cells still covered with 10 mL PBS-EDTA and re-incubate them for 1 - 3 min at 37 &#176;C. Stop the enzymatic reaction by adding 1 mL FCS and collect the detached cells in a 50-mL conical tube. Centrifuge the cells at 200 x g for 5 min, resuspend the pellet in keratinocyte medium, and seed 6 x 105 cells into a fresh T75 cell culture flask. 
	NOTE: To generate organotypic skin reconstructs, the primary keratinocytes should be used no later than passage 3 - 4.</li>
</ol>
4. Isolation of Primary Fibroblasts from the Dermis
<ol>
	<li>Cut the dermal tissue (section 1.3) into smaller pieces (1 mm2) and transfer them into a 50-mL conical tube. Add 10 mL collagenase-solution (5 U/mL in PBS+) and incubate for 45 min in a water bath at 37 &#176;C. Centrifuge the mixture of tissue pieces and cells at 200 x g for 5 min.</li>
	<li>Wash the cell pellet twice with 10 mL DMEM containing 4.5 g/L glucose and L-Glutamine, but without L-Pyruvate. Finally resuspend the tissue pieces in 2 mL of DMEM containing 1% (v/v) gentamycin and 10% FCS. Transfer them into a T25 cell culture flask and incubate them in a cell incubator at 37 &#176;C and 5% CO2 overnight. 
	NOTE: The small volume allows the fibroblasts to migrate out of the tissue-debris and forces them to attach to the culture flask.</li>
	<li>The next morning, add another 6 mL DMEM/Gentamycin/FCS and incubate for 2 - 3 days at 37 &#176;C until the cells have reached 80 - 90% confluency.</li>
</ol>
5. Cultivation of Primary Fibroblasts
<ol>
	<li>To passage the fibroblasts, remove the medium and wash adherent cells with PBS. Add 5 mL 1x Trypsin-EDTA and incubate for 3 min at 37 &#176;C.</li>
	<li>Stop the enzymatic reaction by adding 5 mL DMEM/FCS and collect the detached cells in a 50-mL conical tube. Centrifuge the cells at 200 x g for 5 min, resuspend the pellet in DMEM medium, and seed 6 x 105 cells into a fresh T75 cell culture flask. 
	NOTE: To generate organotypic skin reconstructs, the primary fibroblasts should be used no later than passage 4 - 6.</li>
</ol>
6. Generation of 3D Melanoma Spheroids Via the Hanging Drop Method
<ol>
	<li>Culture melanoma cells (e.g., 451-LU, or any melanoma cell line of interest) according to general protocols, using RPMI containing 10% FCS15.
	<ol>
		<li>To generate melanoma spheroids of similar size and quality, wash the melanoma cells in PBS, add 5 mL of 1x Trypsin-EDTA in PBS to the cells in a T175 cell culture flask, and incubate for 3 - 5 min at room temperature (RT). Neutralize the Trypsin by adding 5 mL RPMI/10% FCS. Harvest the cells by centrifugation at 200 x g for 5 min. Re-suspend the cell pellet in RPMI/FCS at a final concentration of 10,000 cells/mL as determined by counting in a hemocytometer.</li>
	</ol>
	</li>
	<li>Spot 40 x 25 &#181;L (= 250 cells) of the cell suspension onto the inner surface of the lid of a sterile non-adhesive cell culture dish (&#216; 10 cm) using an electronic multi-pipette. With a fast but smooth movement, turn the lid around and place it on the respective cell culture dish containing 5 mL PBS.
	<ol>
		<li>Culture &#34;hanging drop dishes&#34; in a cell incubator at 37 &#176;C and 5% CO2 for 10 - 14 days, depending on the cell type used. 
		NOTE: Cells from the MM growth phase typically grow faster and form more solid spheroids in the hanging drop compared to melanoma cells derived from the RGP or VGP. For individual evaluation, observe spheroid growth under a pair of binoculars or under a light microscope (4X&#160;magnification). Usually, spheroids become detectable after 48 h under a pair of binoculars or under a light microscope (4X&#160;magnification). After 10 - 14 days, they are visible without any magnification device.</li>
	</ol>
	</li>
	<li>5 days after the initial drop spotting, add 10&#160;&#181;L of fresh RPMI/10% FCS medium to each drop. Subsequently, exchange 10&#160;&#181;L of medium every other day. The use of an electronic dispenser is very helpful in this step.</li>
	<li>Depending on the cell type, harvest the spheroids (see section 10 for details) after 10 - 15 days by gently rinsing them off the cell culture dish lid with PBS. Collect them in a fresh non-adhesive cell culture dish. 
	NOTE: The cultivation period depends on the tumor growth phase of the melanoma cells when they were initially derived, e.g., spheroids derived from 451-LU cells grow to approximately 500 &#181;m in diameter within 12 days of culturing in the hanging drop15.</li>
</ol>
7. Generation of the Dermal Compartment of Organotypic Full Skin Reconstructs
<ol>
	<li>Generate skin models using 24-well cell microporous membrane inserts (pore size 8 &#181;m) placed in 24-well plates. 
	NOTE: Make sure not to use hanging but standing inserts, because they will be placed into a 6-well plate for air-liquid cultivation at a later stage.</li>
	<li>Prepare the gel neutralization solution (GNL)15, as well as the culture media referred to as MM and endothelial growth media (EGM)16,17. To prevent coagulation, store the collagen type I (usually from rat tail, 3.5 - 4 mg/mL in 0.02 N acetic acid) on ice until use, because it starts to gel at RT.</li>
	<li>Re-suspend 1 x 105 fibroblasts per insert in GNL and quickly mix the cell suspension with collagen at a ratio of 1:3 in a final volume of 500 &#181;L/insert. Mix by gently pipetting up and down to avoid bubble formation as bubbles may impair the quality of the skin reconstruct.</li>
	<li>Allow the individual dermal gels to settle by keeping them without medium at RT for 30 min in a sterile hood. Subsequently cover each gel with DMEM containing 4.5 g/L glucose, 1% L-glutamine, 10% FCS, and without L-pyruvate, and incubate overnight at 37 &#176;C.</li>
</ol>
8. Generation of the Epidermal Compartment of Organotypic Full Skin Reconstructs
<ol>
	<li>The next day remove the medium from the dermal gels and equilibrate them with EGM media (10% FCS, 1% PenStrep, 10 mg/mL gentamicin) for 2 h at 37 &#176;C. Withdraw the medium and carefully seed 1 x 105 keratinocytes resuspended in 100 &#181;L EGM on top of the dermal gel.</li>
	<li>Incubate the reconstructs for 1.5 h at 37 &#176;C to allow the keratinocytes to adhere to the dermal compartment. 
	NOTE: During this incubation time, the gels will start to shrink due to fibroblast-induced contraction, and thus, at least partially detach from the insert walls.</li>
	<li>Cover the skin equivalents with approximately 800 &#181;L EGM and carefully remove the residual gel from the insert wall with a small (white) pipette tip.Culture the skin equivalents submerged in EGM for 7 days in a cell incubator at 37 &#176;C, 5% CO2, and change the medium every other day. 
	NOTE: During this time the skin equivalents will shrink significantly.</li>
</ol>
9. Air-liquid Cultivation of Organotypic Full Skin Reconstructs
<ol>
	<li>At day 8, transfer each insert into an individual well of a 6-well plate. Only add 1.2 - 1.4 mL of MM medium to each well, so that the skin reconstruct is supplied with medium from the bottom of the well but is not covered with medium. 
	NOTE: Cultivation at the air-liquid interface allows stratification of the epidermal part and establishment of a full cornified layer (stratum corneum).</li>
	<li>During the next 10 - 17 days, change the medium as stated in section 9.1 every other day. During this period, add drugs or other stimuli as needed to the medium. Carefully remove the full skin reconstruct from the microporous membrane insert using curved tweezers for further analysis, e.g., immunohistochemical analysis (Figure 1).</li>
</ol>
10. Generation of the Organotypic Melanoma Spheroid Skin Models
<ol>
	<li>To integrate the melanoma spheroids into the dermal compartment of the organotypic full skin reconstructs, carefully rinse the spheroids (after step 6.4) off the lid of the hanging drop cell culture dish with PBS. Collect 10 - 20 spheroids/insert in a sterile non-adhesive cell culture dish. Carefully remove excessive PBS with a Pasteur pipette.</li>
	<li>Aspirate the spheroids in the smallest volume possible of EGM. At this point observe and count the spheroids with the naked eye, without any further magnification device. Take 10 - 20 spheroids per skin model/gel, as stated in step 10.1.</li>
	<li>Transfer them to the desired volume of GNL containing fibroblasts (during step 7.3), and mix with the collagen type I. From this step on proceed as described in sections 7.3 - 9.1. 
	NOTE: Tumor spheroids become visible in the dermal gel as white spots (Figure 2)</li>
</ol>

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The authors declare that they have no competing financial interests.

The organotypic melanoma spheroid skin model introduced here warrants new insights for a deeper understanding of intra-tumoral and tumor-host interaction, and may provide an advanced screening platform to study molecular mechanisms of tumor development and therapy resistance in the future.
To guarantee the best physiologic and in vivo mimicking conditions of skin reconstructs, the quality of the primary cells is of utmost importance. As stated above, juvenile or pre-natal skin cells show the lowest differentiation grade and are therefore best suitable to generate full-thickness skin equivalents. The first possible quality control is the extent of dermal contraction at day 2 after seeding the keratinocytes and equilibrating the gel to EGM (Protocol sections 8.2 and 8.3). Dermal gels will shrink the most with the best quality of fibroblasts. Also, the integration of too few or too many fibroblasts may impair dermal contraction and consequently attachment of the epidermal to the dermal compartment. This in turn will compromise epidermal differentiation, because this process requires an extensive cross-talk between dermal and epidermal cells.
The quality of primary cells also critically depends on the cell confluency as well as the passage of the cells. If keratinocytes are grown to &#8805;80% confluency they immediately stop proliferating and start to differentiate. It is therefore important to culture them to 40 - 70% confluency during passaging and use them no later than passage 3 - 4. Fibroblasts are less sensitive but should not be used later than passage 4 - 6. Please also note that all primary cells and cell lines used to generate organotypic melanoma spheroid skin models are cultured free of antibiotics, and therefore the contamination risk is high. However, the addition of antibiotics changes the physiology of the individual cells and therefore reduces the quality of the skin equivalents.
In general, tumor spheroid formation is possible from almost all kinds of tumor cell lines and also from tumor cells freshly isolated from patient material. The initial cell number and/or cultivation time for gaining optimal spheroid sizes may vary depending on the cell type used. Up to a size of 150 - 200 &#181;m, all cells included in a spheroid can still be sufficiently supplied with nutrients via simple diffusion. Only spheroids with sizes &#8805;500 &#181;m show a high degree of cellular differentiation and represent typical features of non-vascularized tumor tissue22.
The organotypic melanoma-spheroid-skin-model developed here is particularly suitable to study tumor-host interactions and for melanoma drug testing under in vivo-like conditions15. Still, the environment of melanoma in vivo is even more complex, harboring a variety of tumor associated cell types, including immune cells and endothelial cells. Since the addition of proper primary immune cells faces problems with histocompatibility, these models are not yet able to adequately monitor immune-therapeutic approaches.
Nevertheless, melanoma spheroids can be generated from freshly isolated patient material and once included into the full skin reconstruct, may serve as individual drug screening platforms. Even the integration of pieces of melanoma metastasis into skin reconstructs is conceivable to test drug combinations in a tailored therapeutic approach. Including the organotypic 3D skin-melanoma model in preclinical testing is likely to help ensure that only the most promising novel therapeutic concepts are taken forward into clinical testing, thus reducing the attrition rate of potential new treatments for this disease and increasing the rate of therapeutic success in clinical trials.

The human skin is composed of two distinct compartments that serve different functions in protecting the body from adverse environmental effects1. The lower dermal compartment consists of a fibro-elastic connective tissue. It is composed of loosely connected collagen and elastin fibers synthesized by fibroblasts, serving a mechanical barrier function. The dermis is separated from the upper epidermis by the basal lamina which is produced as an extracellular matrix due to a constant communication between both skin compartments. In contrast to the dermis, the epidermis is a squamous epithelium which mainly consists of keratinocytes and can be differentiated into four layers. The stratum basale consists of undifferentiated basal keratinocytes which constantly derive from skin progenitor cells stratifying through the stages of the stratum spinosum and stratum granulosum into the stratum corneum to protect the body from dehydration and infections2. Melanocytes are aligned at the basal membrane, and communicate through dendritic extensions with multiple keratinocytes. They produce the pigment melanin to protect the skin tissue from the adverse effects of UV radiation, like skin ageing, immunosuppression, inflammation, and induction of non-melanoma skin cancer. UV radiations contribution to the transformation of melanocytes to malignant melanoma, however, is still under debate3.
Melanoma development is differentiated into different tumor progression stages, and characterized by certain genetic, morphologic, and histologic changes4. They originate either de novo or from an innate or acquired nevus due to a local increase of melanocyte proliferation causing benign neoplasia. This precursor lesion may convert into structurally modified dysplastic tissue containing atypic cells, which may continue to the first malignant stage, the radial growth phase (RGP). This early tumor progression phase is characterized by cells radially proliferating within the epidermis, showing few locally invasive cells within the papillary dermis. During the subsequent vertical growth phase (VGP) melanoma cells already show a metastatic and invasive phenotype by breaking through the basal lamina to infiltrate the deeper parts of the dermis as well as the subcutaneous tissue5. Finally, the metastatic melanoma (MM) represents the most aggressive progression stage with metastatic cells systemically spreading throughout the blood- and lymph system to invade distally organs like liver, lung, and brain6.
To date, early diagnosis followed by surgery still remains the most effective therapy of malignant melanoma. The prognosis for patients with distant metastasis, however, remains particularly poor7, because classical chemotherapy regimens confer only little survival benefit8,9. However, after decades of stagnation, recent advances in targeted therapies have considerably improved the prognosis of malignant melanoma.
Dysregulation of two major mitogen activated pathways, the RAS-RAF-MEK-ERK and the PI3K-AKT-PTEN signaling pathways, present key drivers of melanoma progression, especially when constitutively activating point mutations of the proto-oncogenes BRAFV600 and NRAS are present10. Accordingly, the invention of targeted kinase inhibitors promised therapeutic benefit for patients suffering from metastatic melanoma. A multitude of clinical trials conducted to this point has not achieved significant benefit for patients with metastatic melanoma. Nearly all responses are partial, with a subpopulation of patients showing primary resistance. Moreover, the acquisition of secondary resistance leading to relapse was observed in the majority of patients11,12.
It becomes clear that analysis of the mutation status alone is not sufficient to develop the most potent therapeutic strategy. New fast and reliable diagnostic tools are necessary to systematically record and analyze the responsiveness of individual cancer cells. The vast majority of currently available data on human melanoma have been obtained from two-dimensional (2D) melanoma cell cultures. Tumor cells, however, grown in 3D allow intercellular crosstalk between differentiated cancer cell subpopulations as well as between cancer cells and the non-transformed surrounding host tissue. Therefore, it would be best to reconstruct the 3D environment in which the melanoma developed, to be used as a preclinical screening model13,14.

<table><tbody><tr><td>fetal serum albumin (FCS)</td><td>Thermo Fisher Scientific</td><td>10270106</td><td>add 10 % to cell culture medium</td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25300054</td><td>1X dilute in PBS</td></tr><tr><td>RPMI</td><td>Thermo Fisher Scientific</td><td>61870010</td><td>for cultivation of melanoma cell lines</td></tr><tr><td>DMEM&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>41965062</td><td>for cultivation of primary fibroblasts</td></tr><tr><td>Dispase</td><td>Gibco life technologies&amp;nbsp;</td><td>17105041</td><td>2U/ml, dissolve in PBS</td></tr><tr><td>Collagen type I</td><td>BD Biosciences</td><td>354249</td><td>usually from rat tail, concentration should be 3.5-4 mg/ml</td></tr><tr><td>24-well inserts Nunclon&amp;nbsp;</td><td>Nunc</td><td>140629</td><td>8 &amp;micro;m pore size; use standing, not hanging inserts!</td></tr><tr><td>cell strainer</td><td>BD Falcon</td><td>352360</td><td>yellow</td></tr><tr><td>Gentamycine</td><td>Thermo Fisher Scientific</td><td>15750060</td><td>dissolve in PBS</td></tr><tr><td>Keralife keratincyte medium</td><td>Cell Systems</td><td></td><td>do not add FCS or antibiotics</td></tr><tr><td>Collagenase</td><td>SERVA</td><td>17454</td><td>NB4 standard grade</td></tr><tr><td>juvenile human keratinocytes</td><td>Cell Systems</td><td>FC-0007</td><td>alternative to own preparation from juvenile foreskin</td></tr><tr><td>juvenile human fibroblasts</td><td>Cell Systems</td><td>FC-0001</td><td>alternative to own preparation from juvenile foreskin</td></tr><tr><td>DMEM&lt;br/&gt; [+] 4,5 g/l Glucose,&lt;br/&gt; [+] L- Glutamine,&lt;br/&gt; [-] L- Pyruvate</td><td>Gibco life technologies</td><td>41965</td><td>mix 1:1 with Ham&amp;acute;s F-12 for MM medium;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; mix 3:1 with Ham&amp;acute;s F-12 for EGM medium</td></tr><tr><td>Ham&amp;acute;s F-12 [+] L-Glutamine</td><td>Gibco life technologies</td><td>&amp;nbsp;21765-029</td><td>add to DMEM for the generation of EGM and MM medium (see lane 15)</td></tr><tr><td>EGF&amp;nbsp;</td><td>Gibco life technologies</td><td>&amp;nbsp;13247-051</td><td>add 10 ng/ml only for the generation of EGM medium</td></tr><tr><td>Calcium chloride</td><td>Merck Chemicals</td><td>2381</td><td>add 1.9 mM for the generation of EGM and 3.8 mM for MM medium</td></tr><tr><td>Selenic acid</td><td>Alfa Aesar</td><td>18851</td><td>add 53 nM for the generation of EGM and MM medium</td></tr><tr><td>Insulin</td><td>Lilly</td><td>HI0219 Hum Pen 3ml 100 E.I./ml</td><td>add 5 &amp;micro;g/ml for the generation of EGM and MM medium</td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>E-0135</td><td>add 0.1 mM for the generation of EGM and MM medium</td></tr><tr><td>P-Ethanolamine</td><td>Sigma-Aldrich</td><td>P-0503</td><td>add 0.1 mM for the generation of EGM and MM medium</td></tr><tr><td>Holo-Transferrine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T-0665</td><td>add 5 &amp;micro;g/ml for the generation of EGM and MM medium</td></tr><tr><td>Triiodthyronine</td><td>Sigma-Aldrich</td><td>T-6397</td><td>add 20 pM for the generation of EGM and MM medium</td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H-088816</td><td>add 0.4 &amp;micro;g/ml for the generation of EGM and MM medium</td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P-8783</td><td>add 10 nM only for the generation of EGM medium</td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H-3784</td><td>add 15 mM for the generation of EGM and MM medium</td></tr><tr><td>Serine</td><td>Sigma-Aldrich</td><td>S-4311</td><td>add 1 mM for the generation of EGM and MM medium</td></tr><tr><td>Cholinchloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>C-7017</td><td>add 0.64 mM for the generation of EGM and MM medium</td></tr><tr><td>dFCS</td><td>Sigma-Aldrich</td><td>F-0392 Lot 086K0361</td><td>add 2 % for the generation of EGM and MM medium</td></tr><tr><td>Adenine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A-9795</td><td>add 0.18 mM for the generation of EGM and MM medium</td></tr><tr><td>L-Glutamine</td><td>PromoCell</td><td>C-42209</td><td>add 7.25 mM for the generation of EGM and MM medium</td></tr><tr><td>Strontiumchloride</td><td>Sigma-Aldrich</td><td>255521</td><td>add 1 mM for the generation of EGM and MM medium</td></tr><tr><td>anti cytokeratin 10 antibody</td><td>Dako</td><td>M7002</td><td>1:50</td></tr><tr><td>anti cytokeratin 14 antibody</td><td>Santa Cruz</td><td>sc-53253</td><td>1:200</td></tr><tr><td>&amp;nbsp;anti laminin 5 antibody</td><td>Santa Cruz</td><td>sc-32794</td><td>1:50</td></tr><tr><td>anti filaggrin antibody</td><td>Thermo Fisher Scientific</td><td>MA5-13440</td><td>1:50</td></tr><tr><td>anti involucrin antibody</td><td>Acris Antibodies</td><td>AM33368PU</td><td>1:50</td></tr><tr><td>secondary polyclonal goat anti-mouse IgG FITC labeled</td><td>LifeSpan Biosciences</td><td>LS-C153907</td><td>1:50</td></tr><tr><td>secondary polyclonal goat anti-mouse IgG Cy3 labeled</td><td>Jackson Immuno Research</td><td>115-165-003</td><td>1:1000</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>10236276001</td><td>1:100</td></tr></tbody></table>

a 3d organotypic melanoma spheroid skin model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Successful treatment of melanoma metastasis can be influenced by the cross-talk between tumor cells as well as between tumor and non-transformed host cells. The purpose of developing organotypic models of cancer in vitro is to provide suitable preclinical test systems that recapitulate the 3D organization and complexity of human melanoma in vivo. This allows the study of the therapeutic impact on the tumor within an organotypic environment and the adverse effects on the surrounding primary tissue in parallel.
To develop the best organotypic skin models, the quality of the primary cells is crucial. It is advantageous to use juvenile primary fibroblasts and keratinocytes, because they are typically less differentiated compared to adult primary skin cells. Juvenile skin cells can either be isolated from juvenile foreskin as described in the protocol sections 1 - 5, but can also be purchased from companies as pre-natal primary fibroblasts and keratinocytes. If purchasing, it is necessary to order cells from different donors to avoid donor-specific results, e.g., for drug sensitivity. The whole protocol is displayed as a scheme in Figure 3.
Quality control of 3D full-skin equivalents requires immunohistochemical analysis. A first impression can be obtained by Hematoxylin-Eosin (H&#38;E) staining of paraffin embedded sections (3 &#181;m). Detailed analysis of the quality of epidermal differentiation and formation of the basal lamina between the dermis and epidermis requires immunohistochemical analysis using specific antibodies against an epidermal stratification marker. This allows distinguishing between undifferentiated, highly proliferative cells located close to the basal membrane and highly differentiated and keratinized cells at the stratum corneum through the formation of distinct epidermal layers in between. As shown by immune-histological staining (Figure 4), differentiation of keratinocytes throughout the epidermis could be achieved similar to normal skin: mainly the undifferentiated cells from the lower epidermal layers (stratum basale and stratum spinosum) stain positive for keratin 14, while the more differentiated cells from the supra-basal layers (stratum granulosum and stratum corneum) stain positive for keratin 10 and involucrin. Accordingly, filaggrin staining could only be observed in highly differentiated cells of the stratum corneum. Most importantly, laminin 5 staining reveals that a basal lamina was generated to physiologically connect the epidermal to the dermal compartment of the artificial skin reconstruct. This proves that a communicating organotypic microenvironment has been generated to host melanoma cells or spheroids for physiologic and pathophysiologic analysis.
For the purpose of melanoma drug screening, single melanoma cells can also be integrated into the dermis of full skin equivalents to allow de novo melanoma nest formation18,19. Therefore, melanoma cells are combined with primary fibroblasts at a ratio of 5:1, centrifuged together at 200 x g for 5 min and resuspended in GNL prior to mixing with collagen. As a result, the melanoma cell nests will spontaneously form in the dermal compartment. According to our experience, only cells of the metastatic growth phase form proper nests, compared to melanoma cells of the RGP or VGP15. One major drawback of these types of models is the fact that the number and size of melanoma nests formed cannot be predicted, and may vary between individual skin reconstructs, independently of any treatment. For example, 1,000 cells seeded into the dermal compartment may gain 10 nests consisting of 100 cells or 100 nests consisting of 10 cells each (Figure 5). These biologic variables present with three deficiencies: first, the number and size of melanoma nests formed are unpredictable; second, the metastases in vivo are usually larger than melanoma nests and exhibit a more complex intra-tumoral diversity; and third, due to the limited life-span of tumor-nest models, treatment is initiated early, and consequently rather inhibits tumor outgrowth instead of causing regression of existing tumor nests.
To overcome these limitations organotypic melanoma spheroid skin models can be generated. By culturing 250 metastatic melanoma cells in a hanging drop for 14 days20, spheroids are reproducibly generated consisting of viable melanoma cells presenting a compact structure with a final diameter of approximately 500 &#181;m mimicking non vascularized tumor nodes, micro-metastasis, or inter-capillary micro regions of solid tumors21,22. In general, any melanoma cell line is suitable for the generation of spheroids via the hanging drop method; however, cells derived from more advanced metastatic tumor stages form more solid spheroids compared to cell lines derived from early progression stages, e.g., the RGP.
For some cells, it is advantageous for proper spheroid formation to enhance the viscosity of the hanging drop culture medium. This can be achieved by the addition of 10 - 50% methyl-cellulose to the culture medium. For the methyl-cellulose stock solution, autoclave 1.2 g methyl-cellulose together with a magnetic stir bar in a 100 mL glass bottle. Add 100 mL preheated (60 &#176;C) medium and stir for 20 min at room temperature, and another 1 - 2 h at 4 &#176;C. Centrifuge the stock solution for 2 h at 5,000 x g and store the viscous supernatant at 4 &#176;C until use.
Proper validation of full skin melanoma spheroid models is provided by the fact that a defined number of melanoma spheroids can - at least statistically - be integrated into the dermal fibroblast/collagen I scaffold at day 1 of the skin model construction, allowing them to co-develop during epidermal differentiation for 25 - 27 more days. The yield of spheroids can be analyzed directly after seeding, because spheroids appear as white spots within the transparent dermal gel and can be seen without any magnification device (Figure 2). As a result, a 3D skin model is generated that harbors mature melanoma spheroids, which had been cultured in vitro for a total of approximately 42 days, showing the highest level of intra-tumoral cell differentiation15.
H&#38;E staining of the skin melanoma spheroid model reveals the histological appearance and cellular distribution of melanoma spheroids to be very similar to the one of non-vascularized human melanoma skin metastases in vivo15 (Figure 6). Two subpopulations of melanoma cells are clearly distinguishable under these conditions: a peripheral proliferating subpopulation and a central subpopulation mainly consisting of shrunken, apoptotic or necrotic cells, forming the so-called &#34;necrotic&#34; center. Immunohistochemically living and proliferating subpopulations can be detected using antibodies against the proliferation marker KI-67, whereas cells of the necrotic center can be visualized by TUNEL-staining15. This particular distribution of tumor cell subpopulations is warranted by the spheroid size (&#8805;500 &#181;m), resulting from a lack of nutrients and oxygen in the central part where catabolic waste accumulates. Following the protocol provided here will allow the generation of a reliable and reproducible organotypic human full-thickness skin model with embedded human melanoma spheroids that mimic human melanoma skin metastasis. Applications of this model include drug testing, screening of toxins, influence of cosmetic compounds or laser therapy on melanoma outgrowth, and treatment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57500/57500fig1.jpg" /> 
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 inserts are transferred to 6-wells and cultivated at the air-liquid interface to allow epidermal stratification. <a href="/files/ftp_upload/57500/57500fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57500/57500fig2.jpg" /> 
Figure 2: Melanoma spheroids embedded into the dermal compartment appear as white spots. While preparing the dermal compartment of the 3D skin reconstruct, a defined number of melanoma spheroids can be added to the fibroblast collagen type I mix. Once the dermal gel has settled, melanoma spheroids become visible as white spots.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57500/57500fig3.jpg" /> 
Figure 3: Scheme of 3D organotypic skin model construction. Remove adipose tissue from the skin sample and cut it into smaller pieces. Incubation with dispase solution overnight at 4 &#176;C facilitates the separation of the epidermis from the dermis. Isolated primary fibroblasts and keratinocytes should be cultivated separately and used between passage 4 - 6 and 3 - 4, respectively. Subsequently, the generation of the 3D skin model can proceed as described in the protocol. <a href="/files/ftp_upload/57500/57500fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57500/57500fig4.jpg" /> 
Figure 4: 3D organotypic skin reconstructs show a differentiation level similar to normal human skin. Paraffin sections of skin equivalents (A) compared to normal human skin (B) were stained for the expression of keratins 14 (red: &#955;ex 554 nm; &#955;em 568 nm) and 10, involucrin (green: &#955;ex 490 nm; &#955;em 525 nm), filaggrin (green: &#955;ex 490 nm; &#955;em 525 nm), and laminin 5 (green: &#955;ex 490 nm; &#955;em 525 nm), and analyzed with a confocal fluorescence microscope. Cell nuclei were visualized by DAPI staining (blue: &#955;ex 340 nm; &#955;em 488 nm). Immunohistochemical examination of the 3D full-thickness of skin equivalents revealed proper epidermal stratification forming distinct layers of the epidermis as seen in normal human skin. While cells from the lower epidermal layers stained positive for keratin 14, the more differentiated cells from the supra-basal layer showed keratin 10 and involucrin staining. Highly differentiated cells close to the stratum corneum expressed filaggrin. Laminin 5 staining shows that a basal lamina is generated to physiologically connect the epidermal to the dermal compartment (lowest panel). This figure has been taken from Voersmann et al.15 with permission. <a href="/files/ftp_upload/57500/57500fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57500/57500fig5.jpg" /> 
Figure 5: The number and size of spontaneously formed melanoma nests cannot be predicted. De novo melanoma nest formation in the dermal compartment of full skin equivalents can be achieved by mixing a defined number of melanoma cells with primary fibroblasts to embed both cell types in the collagen type I matrix. The number and size of spontaneously formed melanoma nests can only be analyzed from a mature 3D skin reconstruct after about 21 days. As depicted from two samples (A) and (B), the numbers and sizes of the melanoma nests may vary between individual skin reconstructs. As a consequence, it is difficult to validate these models and to predict the therapeutic impact. <a href="/files/ftp_upload/57500/57500fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57500/57500fig6.jpg" /> 
Figure 6: Melanoma spheroids integrated into skin equivalents recapitulate key features of human cutaneous melanoma metastasis. H&#38;E stained paraffin sections of tumor spheroids embedded into skin equivalents revealed spheroids to share key features with non-vascularized human cutaneous melanoma metastases in vivo. Two subpopulations of cells are clearly discernible: a peripheral living subpopulation and central subpopulation mainly consisting of shrunken, apoptotic or necrotic cells, forming the &#34;necrotic&#34; center. This distribution of tumor cell subpopulations is guaranteed by the spheroid size. This figure has been modified from Voersmann et al.15 with permission. <a href="/files/ftp_upload/57500/57500fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A 3D Organotypic Melanoma Spheroid Skin Model at JoVE.com

A 3D Organotypic Melanoma Spheroid Skin Model

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

a-3d-organotypic-melanoma-spheroid-skin-model

Research

JoVE Journal

Cancer Research

15.6K Views.  TU-Dresden. Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Video: A 3D Organotypic Melanoma Spheroid Skin Model

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Bioengineering

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Patient-Derived Melanoma Organoids: A Frontier in Cancer Immunotherapy

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While traditional two- and three-dimensional models can offer insights into cancer development and progression, these lack crucial aspects that hinder a faithful mimic of native tumors. An alternative model that has gained a lot of attention is the patient-derived organoid. The development of these organoids recapitulates the complex intercellular communication, tumor microenvironment, and histoarchitecture of tumors. This paper describes the protocol for establishing melanoma patient-derived organoid (MPDO) models. To validate these models, we assessed the immune cell composition, including the expression levels of T-cell activation markers, to confirm the cellular heterogeneity of the organoids. Additionally, to describe the potential utility of MPDOs in cellular therapies, we evaluated the cytotoxic capabilities of treating the organoids with &#947;&#948; T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

Author Spotlight: Recreating Melanoma Complexity with Patient-Derived Organoids for Immunotherapy Evaluation

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Medicine

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Efficient 3D Skin Models for Flexible Research Application

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

Author Spotlight: Enhancing Skin Model Diversity with Cost-Effective 3D Cellular Models

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

Patient-derived Xenograft Modeling: A Technique to Generate Melanoma Mouse Models

Source: Min Xiao et al. <a href="https://www.jove.com/v/59508"> A Melanoma Patient-Derived Xenograft Model</a>. J. Vis. Exp. (2018)
In this video, human-derived melanoma cells are implanted subcutaneously into the flank region of an immunocompromised mouse. The PDX model allows preclinical investigation of melanoma cells that better recapitulate the tumor heterogeneity and melanoma aggressiveness observed in in vivo conditions.

Modelowanie ksenoprzeszczepów pochodzących od pacjenta: technika generowania mysich modeli czerniaka

Source: Min Xiao et al.  A Melanoma Patient-Derived Xenograft Model. J. Vis. Exp. (2018)
In this video, human-derived melanoma cells are implanted ...

JoVE Encyclopedia of Experiments

Skin Cancer

Animal model studies

Hanging Drop Method: A Technique to Generate 3D Melanoma Spheroids

Source: M&#252;ller, I. et al., <a href="https://www.jove.com/v/57500"> A 3D Organotypic Melanoma Spheroid Skin Model</a>. J. Vis. Exp. (2018)
This video describes the technique of generating three-dimensional spheroids via the hanging drop method using melanoma cells. The generated in vitro&#160;three-dimensional (3D) organotypic melanoma spheroid model&#160;can portray the&#160;in vivo&#160;architecture of malignant melanoma and may give insights into intra-tumoral interactions.&#160;

Metoda wiszącej kropli: technika generowania sferoid czerniaka 3D

Source: M&#252;ller, I. et al.,  A 3D Organotypic Melanoma Spheroid Skin Model. J. Vis. Exp. (2018)
This video describes the technique of generating ...

A 3D Organotypic Melanoma Spheroid Skin Model

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The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression

A Melanoma Patient-Derived Xenograft Model

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Modelowanie ksenoprzeszczepów pochodzących od pacjenta: technika generowania mysich modeli czerniaka

Metoda wiszącej kropli: technika generowania sferoid czerniaka 3D