Name: generation of self-assembled vascularized human skin equivalents
Uploaded: 2021-02-12T14:45:00.000+00:00
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><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&#039;s Modification of Eagle&#039;s Medium) with 4.5 g/L glucose, L-glutamine &amp;amp; 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&#039;s Mod. Of Eagle&#039;s Medium/Ham&#039;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&quot;, 5/32&quot;, 3/16&quot;, 7/32&quot;, 1/4&quot;, 9/32&quot;, 5/16&quot;, 3/4&quot;, 7/16&quot;, 1/2&quot;, 5/8:, 3/4&quot;. 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&quot; or 1/2&quot;.</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.&amp;nbsp;</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 colspan="4">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 &amp;amp; 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 &amp;amp; 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 &amp;micro;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,&amp;nbsp;</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

6.3K 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

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

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

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

Developmental Biology

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

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 ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

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 ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

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 ...

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 ...

Generation of Self-assembled Vascularized Human Skin Equivalents

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Chapters in this video

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

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

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

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

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

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

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

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