Name: generation of self-assembled vascularized human skin equivalents
Uploaded: 2021-02-12T14:45:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Generation of Self-assembled Vascularized Human Skin Equivalents at JoVE.com

The authors thank Dr. Jim Rheinwald59 and Dr. Ellen H. van den Bogaard20 for their generous gift of N/TERT cell lines. This work was supported by the American Heart Association (19IPLOI34760636).

The authors have nothing to disclose.

This protocol has demonstrated a simple and repeatable method for the generation of VHSEs and their three-dimensional analysis. Importantly, this method relies on few specialized techniques or equipment pieces, making it accessible for a range of labs. Further, cell types can be replaced with limited changes in the protocol, allowing researchers to adapt this protocol to their specific needs.
Proper collagen gelation is a challenging step in establishing skin culture. Especially when using crude preparations without purification, trace contaminants could influence the gelation process. To help ensure consistency, groups of experiments should be performed with the same collagen stock that will be used for VHSE generation. Further, the gelation should ideally occur at a pH of 7-7.4, and trace contaminants may shift the pH. Before using any collagen stock, a practice acellular gel should be made at the desired concentration and the pH should be measured prior to gelation. Completing this collagen quality check before beginning dermal component seeding will identify the problems with proper gelation and collagen homogeneity prior to setting up a complete experiment. Instead of seeding acellular collagen directly onto a culture insert, seed some collagen onto a pH strip that evaluates the whole pH scale and verify a pH of 7-7.4. Gelation can be evaluated by applying a droplet of the collagen gel solution onto a coverslip or tissue culture plastic well plate (a well plate is recommended to simulate the confined sides of a culture insert). After gelation time, the collagen should be solid, i.e., it should not flow when the plate is tilted. Under phase contrast microscopy, the collagen should look homogeneous and clear. Occasional bubbles from collagen seeding are normal but large amorphous blobs of opaque collagen within the clear gel indicates a problem-likely due to insufficient mixing, wrong pH, and/or failure to keep the collagen chilled during mixing.
The cell seeding amounts and media may be adjusted. In the protocol above, the encapsulated cell amounts have been optimized for a 12-well insert at 7.5 x 104 fibroblasts and 7.5 x 105 endothelial cells per mL of collagen with 1.7 x 105 keratinocytes seeded on top of the dermal construct. Cell densities have been optimized for this VHSE protocol based on the preliminary studies and the previous research investigating 3D vascular network generation in various collagen concentrations48 and HSE generation22,80,81. In similar systems, the published endothelial cell densities are 1.0 x 106 cells/mL collagen48; the fibroblast concentrations often range from 0.4 x 105 cells/mL of collagen22,28,82 to 1 x 105 cells/mL of collagen8,58,83,84,85; and the keratinocyte concentrations range from 0.5 x 105 [cells/cm2]80 to 1 x 105 [cells/cm2]8. Cell densities can be optimized for specific cells and research question. Three-dimensional cultures with contractile cells, such as fibroblasts, can contract leading to viability reduction and culture loss86,87. Preliminary experiments should be completed to test contraction of the dermal compartment (which can occur with more dermal cells, more contractile dermal cells, longer submersion cultures, or softer matrices) and to test epidermal surface coverage. Additionally, the number of days in submersion and the rate of tapering the serum content can also be customized if excessive dermal contraction is occurring or a different rate of keratinocyte coverage is required. For example, if contraction is noticed during the period of dermal submersion or while keratinocytes are establishing a surface monolayer, moving more quickly through the serum tapering process and raising VHSEs to ALI can aid in preventing additional contraction. Similarly, if keratinocyte coverage is not ideal, changing the number of days that the VHSE is submerged without serum may help increase the epidermal monolayer coverage and mitigate the contraction since serum is left out. Changes in cell densities or other suggestions above must be optimized for the specific cultures and research goals.
To establish a proper stratification of the epidermis during the air liquid interface (ALI) period, it is critical to regularly check and maintain fluid levels in each well so that ALI and appropriate hydration of each construct is kept throughout the culture length. Media levels should be checked and tracked daily until consistent ALI levels are established. The epidermal layer should look hydrated, not dry, but there should not be pools of media on the construct. During ALI, the construct will develop an opaque white/yellow color which is normal. The epidermal layer will likely develop unevenly. Commonly, the VHSEs are tilted due to the collagen seeding or dermal contraction. It is also normal to observe a higher epidermal portion in the middle of the construct in smaller constructs (24 well size) and a ridge formation around the perimeter of the VHSE in 12 well size. Contraction of the constructs13 may change these topographical formations, and/or may not be observed at all.
Staining and imaging of VHSEs introduces mechanical manipulation to the VHSEs. It is very important to plan and limit manipulation of each culture. When manipulation is necessary, maintain gentle movements when removing VHSEs from the insert membranes, when adding staining or wash solutions to the construct surface, and when removing and replacing VHSEs in their storage/imaging wells during imaging preparation. Specifically, the apical layers of the epidermal component may be fragile and are at risk of sloughing off the basal epidermal layers. Apical layers of the epidermis are fragile and go through desquamation even in native tissue4, but for accurate analysis of epidermal structure it is important to minimize damage or loss. If epidermal layers lift off the construct, they can be imaged separately. The basal layers of the epidermis are most likely still attached to the dermis while portions of the apical layers may detach. For visualization of the epidermis, a nuclear stain is helpful in observing this since dense nuclei is a characteristic of lower and mid layers of the epidermis.
Confocal imaging of the VHSE post-fixation has been discussed in the protocol, but it is also possible to image the VHSEs throughout the culture via upright based optical coherence tomography (OCT)88,89,90,91,92,93. VHSE are stable enough to withstand imaging without incubation or humidification for at least two hours without noticeable effects. As OCT is label free and noninvasive, it is possible to track the epidermal thickness during maturation. Other noninvasive imaging modalities can likely be employed as well.
Volumetric imaging of the combined dermal and epidermal structures can be challenging due to laser attenuation deeper in the VHSE. This can be mitigated by imaging the construct in two orientations, from the epidermal side (Figure 1) and from the dermal side (Figure 2), allowing for good resolution of dermal vascular structures and the epidermis. Additionally, the sample can be cleared, allowing for volumetric images of the entire structure with minimal attenuation. Several clearing methods were attempted, however, the methanol/methyl salicylate method described yielded the best results. Researchers interested in optimizing other clearing methods are directed towards these reviews49,61,62. If clearing, it is suggested to fully image the sample prior to clearing, as the method can damage the fluorophores and/or the structure. Further, the imaging should be completed as soon as possible after clearing, as the fluorescence may fade within days.
For simplicity and accessibility, this protocol utilized the simplest media blends found in previous literature11,80. Although there are many advantages to using simple media blends, the limitations of this choice are also recognized. Other groups have studied the effects of specific media components on epidermal and dermal health and found that other media additives94, such as external free fatty acids/lipids, enhance the stratum corneum of the epidermis and improve the skin barrier function. Although our immunofluorescent markers show appropriate differentiation and stratification in the epidermis, depending on the studies being conducted, additional media optimization may be needed. Further, an extensive analysis of the epidermal BM was not conducted when evaluating the VHSEs presented here. The integrity of the BM is an important indication of skin equivalents; various groups have done research on the culture duration and its effect on BM markings95 as well as analysis of fibroblast presence and added growth factor effects on BM expression14. It is important to note that analysis of the BM component should be evaluated and optimized when using this protocol.
In this protocol is described a procedure for VHSE generation, demonstrating results after 8 weeks at ALI. VHSE cultures have been cultured up to 12 weeks at ALI without noticeable change or loss of viability, and it is possible that they may be viable longer. Importantly, this protocol is readily adaptable to commonly available cell types, as demonstrated by the replacement of dermal fibroblasts with IMR90 lung fibroblasts. Depending on the researcher&#39;s need and available resources, the cell types and media blends on the culture can be adjusted, although more dissimilar cell types may require media optimization. In summary, these procedures are meant to provide clarity on the culture of VHSEs for the study of skin biology and disease. To maximize accessibility, the protocol was developed this simple and robust using common equipment, cell lines, and reagents as a minimal effective approach that can be further customized to the specific needs of research studies.

Human skin performs many essential biological functions including acting as an immune/mechanical barrier, regulating body temperature, participating in water retention and sensory roles1,2,3,4. Anatomically, skin is the largest organ in the human body and is made up of three main layers (epidermis, dermis, and hypodermis) and possesses a complex system of stromal, vascular, glandular, and immune/nervous system components in addition to epidermal cells. The epidermis itself is composed of four layers of cells that are continuously renewed to maintain barrier function and other structures of native skin (i.e., sweat and sebaceous glands, nails)3. Skin physiology is important in immune function, wound healing, cancer biology, and other fields, leading researchers to use a wide range of models, from in vitro monocultures to in vivo animal models. Animal models offer the ability to study the full complexity of skin physiology, however, commonly used animal models such as mice have significant physiological differences when compared to humans5. These limitations, and the increased cost of animal models, have led many researchers to focus on developing in vitro models that more closely reflect the physiology of human skin1,6. Of these, one of the simpler model types is the human epidermal equivalent (HEE; also referred to as half-thickness skin models) which are composed of only epidermal keratinocytes on an acellular dermal matrix, but capture epidermal differentiation and stratification seen in vivo. Building on this, models containing dermal and epidermal components (keratinocytes and fibroblasts) are often referred to as human skin equivalents (HSE), full-thickness skin models, or organotypic skin constructs (OSC). Briefly, these models are generated by encapsulating dermal cells within gel matrices and seeding epidermal cells on top. Epidermal differentiation and stratification can then be achieved via specialized media and air exposure7. Skin equivalents have most often been generated through self-assembly techniques using dermal gels made of collagen type I (either of rat tail or bovine skin origin)1,8, but similar models have incorporated other matrix components such as fibrin9,10, fibroblast derived11,12, cadaveric de-epidermized membranes13,14,15,16, commercially available gels and others1,12,13,17,18,19. Currently, there are skin equivalents commercially available (as previously reviewed1,2). However, these are primarily developed for therapeutic purposes and cannot be readily customized to specific research questions.
HSEs have been applied in studies of wound healing, grafting, toxicology, and skin disease/developement11,12,13,16,8,20,21,22,23. Although 3D culture more comprehensively models functions of human tissue compared to 2D cultures24, the inclusion of diverse cell types that more accurately reflect the in vivo population enables studies of cell-cell coordination in complex tissues24,25,26. Most HSEs only include dermal fibroblasts and epidermal keratinocytes27, although the in vivo&#160;skin environment includes many other cell types. Recent studies have started including more cell populations; these include endothelial cells in vasculature10,28,29,30,31,32,33,34, adipocytes in sub-cutaneous tissue35,36, nerve components19,21, stem cells27,37,38, immune cells10,39,40,41,42, and other disease/cancer specific models16, 40, 43,44,45,46,47. Particularly important among these is vasculature; while some HSEs include vascular cells, overall they still lack comprehensive capillary elements with connectivity across the entire dermis10,29 , extended in vitro&#160;stability28, and appropriate vessel density. Further, HSE models are typically assessed through post-culture histological sectioning which limits analysis of the three-dimensional structure of HSEs. Three dimensional analysis allows for volumetric assessment of vascular density48,49 as well as regional variation of epidermal thickness and differentiation.
Although HSEs are one of the most common organotypic models, there are many technical challenges in generating these constructs including identification of appropriate extracellular matrix and cell densities, media recipes, proper air liquid interface procedures, and post-culture analysis. Further, while HEE and HSE models have published protocols, a detailed protocol incorporating dermal vasculature and volumetric imaging rather than histological analysis does not exist. This work presents an accessible protocol for the culture of vascularized human skin equivalents (VHSE) from mainly commercial cell lines. This protocol is written to be readily customizable, allowing for straight-forward adaptation to different cell types and research needs. In the interest of accessibility, availability, and cost, the use of simple products and generation techniques was prioritized over the use of commercially available products. Further, straightforward volumetric imaging and quantification methods are described that allow for assessment of the three-dimensional structure of the VHSE. Translating this procedure into a robust and accessible protocol enables non-specialist researchers to apply these important models in personalized medicine, vascularized tissue engineering, graft development, and drug evaluation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 N NaOH</td>
			<td>Fisher Chemical</td>
			<td>S318-100</td>
			<td>(Dilute from Lab stock)</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>ACROS Organics</td>
			<td>#41678-5000, Lot # B0143461</td>
			<td>Made up using solid Paraformaldehyde in PBS, pH adjusted to 6.9</td>
		</tr>
		<tr>
			<td>Autoclaved forceps</td>
			<td>Fine Science Tools</td>
			<td>#11295-00</td>
			<td>Dumont #5 forceps</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher bioreagents</td>
			<td>Cat # BP510-250, Lot # 190231</td>
			<td>Rnase, Dnase, Protease-Free</td>
		</tr>
		<tr>
			<td>Cell line, Endothelial: Microvascular Endothelial Cell (HMEC1)</td>
			<td>ATCC</td>
			<td>CRL-3243</td>
			<td>SV40 Immortalized microvascular endothelial cell. Note that 750,000 cells/mL of collagen were used.</td>
		</tr>
		<tr>
			<td>Cell line, Fibroblasts: dermal Human fibroblast, adult</td>
			<td>ATCC</td>
			<td>PCS-201-012</td>
			<td>Primary dermal cells. Note that 75,000 cells/mL of collagen were used.</td>
		</tr>
		<tr>
			<td>Cell line, Fibroblasts: human lung firbroblast (IMR90)</td>
			<td>ATCC</td>
			<td>CCL-186</td>
			<td>Primary embryonic cells. Note that 75,000 cells/mL of collagen were used.</td>
		</tr>
		<tr>
			<td>Cell line, Keratinocyte: N/TERT-1</td>
			<td></td>
			<td></td>
			<td>Immortalized via hTERT expression. N/TERT-1 was made using a retroviral vector conferring hygromycin resistance. Cell line established by Dickson et al. 2000. Can be replaced with ATCC PCS-200-010 or PCS-200-011. Note that 170,000 cells were used per construct; N/TERT1 cells must be used from plates that are 30% confluent- two 30% confluent 90 mm tissue culture dishes give more than enough cells.The authors thank Dr. Jim Rheinwald and Dr. Ellen H. van den Bogaard for their generous gift of N/TERT cell lines.</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific; Sorvall Legend X1R</td>
			<td></td>
			<td>(standard lab equipment)</td>
		</tr>
		<tr>
			<td>Computational Software</td>
			<td>MATLAB</td>
			<td>MATLAB 2020a</td>
			<td>MathWorks, Natick, MA.</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica TCS SPEII confocal</td>
			<td></td>
			<td>Laser scanning confocal. Can be replaced with other confocals or deconvolution microscopy.</td>
		</tr>
		<tr>
			<td>Cover Glass (22 x 22)</td>
			<td>Fisher Scientific</td>
			<td>12-545F</td>
			<td>0.13-0.17 mm No.1 Thickness</td>
		</tr>
		<tr>
			<td>Cyanoacrylate super glue or silicone grease</td>
			<td>Glue Masters</td>
			<td>#THI0102</td>
			<td>Glue Masters, THICK, Instant Glue, Cyanoacrylate; super glue is preferred</td>
		</tr>
		<tr>
			<td>DMEM media base</td>
			<td>Corning; Mediatech, Inc</td>
			<td>REF # 10-013-CM; Lot # 26119007</td>
			<td>DMEM, 1X (Dulbecco&#39;s Modification of Eagle&#39;s Medium) with 4.5 g/L glucose, L-glutamine &#38; sodium pyruvate</td>
		</tr>
		<tr>
			<td>DMEM/F-12 50/50</td>
			<td>Corning; Mediatech, Inc</td>
			<td>REF # 10-090-CV; Lot # 21119006</td>
			<td>DMEM/F-12 50/50, 1X (Dulbecco&#39;s Mod. Of Eagle&#39;s Medium/Ham&#39;s F12 50/50 Mix) with L-glutamine</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>#V1101</td>
			<td>(standard lab reagent)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>Cat # FB12999102, Lot # AE29451050</td>
			<td>Research Grade Fetal Bovine Serum, Triple 0.1 um sterile filtered</td>
		</tr>
		<tr>
			<td>Fine tip forceps</td>
			<td>Fine Science Tools</td>
			<td>#11295-00</td>
			<td>Dumont #5 forceps</td>
		</tr>
		<tr>
			<td>Human Epidermal Growth Factor (EGF)</td>
			<td>Peprotech</td>
			<td>Cat # AF-100-15-1MG, Lot # 0318AFC05 D0218</td>
			<td>Made up in 0.1% BSA in PBS</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Alpha Easar</td>
			<td>Lot # 5002F2A</td>
			<td>made up in DMSO</td>
		</tr>
		<tr>
			<td>Insulin (human)</td>
			<td>Peprotech</td>
			<td>Lot # 9352621</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Light/Phase Contrast Microscope</td>
			<td>VWR</td>
			<td>76317-470</td>
			<td>(standard lab equipment)</td>
		</tr>
		<tr>
			<td>Isoproterenol</td>
			<td>Alfa Aesar</td>
			<td>#AAJ6178806</td>
			<td>DL-Isoproterenol hydrochloride, 98%</td>
		</tr>
		<tr>
			<td>Keratinocyte-SFM (1x) media base</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>REF #: 10724-011; Lot # 2085518</td>
			<td>Keratinocyte-SFM (1X); serum free medium</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid</td>
			<td>Fisher Chemical</td>
			<td>Cat # A61-100, Lot # 181977, CAS # 50-81-7</td>
			<td>Crystalline. L-Ascorbic acid can also be purchased as a salt</td>
		</tr>
		<tr>
			<td>L-glutamine (solid)</td>
			<td>Fisher Bioreagents</td>
			<td>CAT # BP379-100, LOT # 172183, CAS # 56-85-9</td>
			<td>L-Glutamine, white crystals or Crystalline powder</td>
		</tr>
		<tr>
			<td>MCDB 131 media base</td>
			<td>Gen Depot</td>
			<td>CM034-050, Lot # 03062021</td>
			<td>MCDB 131 Medium Base, No L-Glutamine, sterile filtered</td>
		</tr>
		<tr>
			<td>Metal punches</td>
			<td>Sona Enterprises (SE)</td>
			<td>791LP, 12PC</td>
			<td>Hollow Leather Punch Set, High Carbon Steel, Hardness: 48HRC; (various sizes including): 1/8&#34;, 5/32&#34;, 3/16&#34;, 7/32&#34;, 1/4&#34;, 9/32&#34;, 5/16&#34;, 3/4&#34;, 7/16&#34;, 1/2&#34;, 5/8:, 3/4&#34;. This punch set is helpful, but x-acto knife can work as well. Size of metal punch that works well for 12 well transwell VHSE is 3/8&#34; or 1/2&#34;.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Chemical</td>
			<td>CAS # 67-56-1</td>
			<td>(optional). For clearing dehydration step.</td>
		</tr>
		<tr>
			<td>Methyl Salicylate</td>
			<td>Fisher Chemical</td>
			<td>O3695-500; Lot # 164535; CAS # 119-36-8</td>
			<td>(optional). For clearing.</td>
		</tr>
		<tr>
			<td>Microtubes, 1.7 mL</td>
			<td>Genesee Scientific Corporation; Olympus Plastics</td>
			<td>Cat # 24-282; Lot # 19467</td>
			<td>1.7 ml Microtubes, Clear; Boilproof, Polypropulene, Certified Rnase, Dnase, DNA, PCR inhibitor and endotoxin-Free</td>
		</tr>
		<tr>
			<td>PBS, 10x Culture grade or autoclaved</td>
			<td>APEX Bioresearch Products</td>
			<td>Cat # 20-134, Lot # 202237</td>
			<td>PBS Buffer, 10x Dry Pack; add contents of pack into container and add water to 1 liter to produce 10x concentrated.</td>
		</tr>
		<tr>
			<td>PBS, 1x Culture grade, (-) Calcium, (-) magnesium</td>
			<td>Genesee Scientific Corporation</td>
			<td>Ref # 25-508; Lot # 07171015</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 1x non-Culture grade</td>
			<td>APEX Bioresearch Products</td>
			<td>Cat # 20-134, Lot # 202237</td>
			<td>PBS Buffer, 10x Dry Pack; add contents of pack into container and add water to 1 liter to produce 10x concentrated. Dilute to 1x with water.&#160;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>Ref # 15140-122, Lot # 2199839</td>
			<td>Pen Strep (10,000 Units/mL Penicillin; 10,000 ug/mL Streptomycin)</td>
		</tr>
		<tr>
			<td>Petri Dish, glass, small</td>
			<td>Corning</td>
			<td>PYREX 316060</td>
			<td>(optional). To be used as a clearing container</td>
		</tr>
		<tr>
			<td>Petri Dishes, 100 mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>Use for making up PDMS.</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>GMID 02065622, Batch # H04719H035</td>
			<td>Sylgard 184 Silicone Elastomer Base, Dow Corning, Midland, MI)</td>
		</tr>
		<tr>
			<td></td>
			<td>Dow Corning</td>
			<td>GMID 02065622, Batch # H047JC4003</td>
			<td>DOWSIL 184, Silicone Elastomer Curing Agent</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>note: PDMS is usually sold as a kit that includes both the base and curing agent components.</td>
		</tr>
		<tr>
			<td>Positive Displacement Pipettes (1000 &#38; 250 uL)</td>
			<td>Gilson</td>
			<td>1000 uL: HM05136, M1000. 250 uL: T12269L</td>
			<td>M1000 pipette capacity (100-1000 uL); M250 pipette capacity to 250 uL</td>
		</tr>
		<tr>
			<td>Positive Displacement Tips/Pistons (1000 &#38; 250 uL)</td>
			<td>Gilson</td>
			<td>1000 uL: CAT # F148180, BATCH # B01292902S; 250 uL: CAT # F148114, BATCH # B05549718S</td>
			<td>Sterilized capillaries and pistons</td>
		</tr>
		<tr>
			<td>Round tipped scoopula</td>
			<td></td>
			<td></td>
			<td>(optional; standard lab equipment) For manipulation of VHSEs prior to imaging</td>
		</tr>
		<tr>
			<td>Supplements for Keratinocyte-SFM media</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>Ref # 37000-015, Lot # 2154180</td>
			<td>Contains EGF Human Recombinant (Cat # 10450-013), Bovine Pituitary Extract (Cat # 13028-014)</td>
		</tr>
		<tr>
			<td>Tissue Culture Plate Inserts, 12 well size, 3 &#181;m pore size</td>
			<td>Corning; Costar</td>
			<td>REF # 3462 - Clear; Lot # 14919057</td>
			<td>Transwell; 12 mm Diameter Inserts, 3.0 um pore size, tissue culture treated, polyester membrane, polystyrene plates,&#160;</td>
		</tr>
		<tr>
			<td>Tissue Culture Plates, 12 well size</td>
			<td>Greiner Bio-one; CellStar</td>
			<td>Cat # 665 180; Lot # E18103QT</td>
			<td>12 well cell culture plate; sterile, with lid; products are sterile, free of detectable Dnase, Rnase, human DNA and pyrogens. Contents non-cytotoxic</td>
		</tr>
		<tr>
			<td>Tissue Culture Plates, 60.8cm^2 growth area</td>
			<td>Genesee Scientific Corporation</td>
			<td>Cat # 25-202; Lot No: 191218-177B</td>
			<td>Tissue culture dishes; treated, growth area 60.8 cm^2; sterile, Dnase, Rnase, Pyrogen Free; virgin polystyrene</td>
		</tr>
		<tr>
			<td>Triton x 100</td>
			<td>Ricca Chemical Company</td>
			<td>Cat # 8698.5-16, Lot # 4708R34</td>
			<td>Wetting agent</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Corning; Mediatech, Inc</td>
			<td>Ref # 25-053-CI</td>
			<td>0.25% Trypsin, 2.21 mM EDTA, 1x [-] sodium bicarbonate</td>
		</tr>
		<tr>
			<td>Type 1 Collagen isolated from Rat tail</td>
			<td>Pel-Freez Biologicals</td>
			<td>56054-1</td>
			<td>Sprauge-Dawley rat tails can be purchased frozen from Pel-Freez or other suppliers. Collagen can be isolated from the tail tendons. Isolation Protocol references [Cross et al.,2010; Rajan et al.,2007; Bornstein, 1958] .Alternatively, high concentration rat-tail collagen can be purchased from suppliers including Corning (Catalog Number: 354249)</td>
		</tr>
		<tr>
			<td>Vaccum chamber, benchtop</td>
			<td>Bel-Art</td>
			<td>F42010-0000</td>
			<td>(standard lab equipment)</td>
		</tr>
		<tr>
			<td>Handheld Precision knife</td>
			<td>X-Acto</td>
			<td>X3311</td>
			<td>(X-Acto knife optional if purchased steel punches)</td>
		</tr>
	</tbody>
</table>

generation of self-assembled vascularized human skin equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

Watch this Scientific Journal Video about Generation of Self-assembled Vascularized Human Skin Equivalents at JoVE.com

Generation of Self-assembled Vascularized Human Skin Equivalents

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Research

JoVE Journal

Bioengineering

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, ...