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 ...

generation-of-self-assembled-vascularized-human-skin-equivalents

Research

JoVE Journal

Bioengineering

6.1K Views.  University of California. 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.

Video: Generation of Self-assembled Vascularized Human Skin Equivalents

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 ...

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 reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

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 supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

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 ...

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 diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

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 applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

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

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 concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

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

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

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 ...

Three-Step Organoid Model for Tendon Research and Engineering

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly arranged collagen type&#160;I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGF&#223; is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Author Spotlight: Advancing Tendon Tissue Engineering with 3D Organoid Models

Herein, we demonstrate a three-step organoid model (two-dimensional [2D] expansion, 2D stimulation, three-dimensional [3D] maturation) offering a promising tool for tendon fundamental research and a potential scaffold-free method for tendon tissue engineering.

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

Methods Article

Generation of Self-assembled Vascularized Human Skin Equivalents