4open 2021, 4, 1Ó A. Idrees et al., Published by EDP Sciences, 2021https://doi.org/10.1051/fopen/2021001Available online at:www.4open-sciences.orgRESEARCH ARTICLEFundamental in vitro 3D human skin equivalent tool developmentfor assessing biological safety and biocompatibility – towardsalternative for animal experimentsAyesha Idrees1,2,3, Inge Schmitz4, Alice Zoso1,5, Dierk Gruhn3, Sandra Pacharra3, Siegfried Shah3,,,Gianluca Ciardelli1, Richard Viebahn2,3, Valeria Chiono1,5,* **, and Jochen Salber2,3,* **1Department of Mechanical and Aerospace Engineering (DIMEAS), Politecnico di Torino, 10129 Turin, ItalyUniversitätsklinikum Knappschaftskrankenhaus Bochum, Department of Surgery, Hospital of the RUHR-University,44892 Bochum, Germany3Department of Experimental Surgery, Centre for Clinical Research, RUHR-University, 44801 Bochum, Germany4Institute of Pathology, RUHR University, 44789 Bochum, Germany5Interuniversity Centre for the Promotion of the 3Rs Principles in Teaching and Research, Centro 3R, 56122 Pisa, Italy2Received 17 December 2020, Accepted 3 February 2021Abstract – Nowadays, human skin constructs (HSCs) are required for biomaterials, pharmaceuticals andcosmetics in vitro testing and for the development of complex skin wound therapeutics. In vitro threedimensional (3D) dermal-epidermal based interfollicular, full-thickness, human skin equivalent (HSE) was heredeveloped, recapitulating skin morphogenesis, epidermal differentiation, ultra-structure, tissue architecture,and barrier function properties of human skin. Different 3D cell culture conditions were tested to optimizeHSE maturation, using various commercially available serum/animal component-free and/or fully deﬁnedmedia, and air-liquid interface (ALI) culture. Optimized culture conditions allowed the production of HSEby culturing normal human dermal ﬁbroblasts (NHDFs) for 5–7 days in CELLnTEC-Prime Fibroblast(CnT-PR-F) medium and then culturing normal human epidermal keratinocytes (NHEKs) for 3 days inCELLnTEC-Prime Epithelial culture (CnT-PR) medium on them. Co-culture was then submerged overnightin CELLnTEC-Prime-3D barrier (CnT-PR-3D) medium to stimulate cell-cell contact formation and ﬁnallyplaced at ALI for 15–20 days using CnT-PR-3D medium. Histological analysis revealed uniform distributionof NHDFs in the dermal layer and their typical elongated morphology with ﬁlopodia. Epidermal compartmentshowed a multi-layered structure, consisting of stratum basale, spinosum, granulosum, and corneum. NHDFsand keratinocytes of basal layer were positive for the proliferation marker Kiel 67 (Ki-67) demonstrating theiractive state of proliferation. The presence of typical epidermal tissue proteins (keratins, laminins, ﬁlaggrin,loricin, involucrin, and b-tubulin) at their correct anatomical position was veriﬁed by immunohistochemistry(IHC). Moreover, transmission electron microscopy (TEM) analyses revealed basement membrane with laminalucida, lamina densa, hemidesmosomes and anchoring ﬁbers. The epidermal layers showed abundant intracellular keratin ﬁlaments, desmosomes, and tight junction between keratinocytes. Scanning electron microscopy(SEM) analyses showed the interwoven network of collagen ﬁbers with embedded NHDFs and adjacentstratiﬁed epidermis up to the stratum corneum similar to native human skin. HSE physiological static contactangle conﬁrmed the barrier function. The developed HSE represents a fundamental in vitro tool to assess biocompatibility of biomaterials, pharmacotoxicity, safety and effectiveness of cosmetics, as well as to investigateskin biology, skin disease pathogenesis, wound healing, and skin infection.Keywords: 3D, Actin, Animal model, Biocompatibility, Biomaterials, Collagen, Culture, Dermal,Dermatoblasts, Development, ECM, Electron microscopy, Extracellular matrix, Effectiveness, Engineering,Epidermal, Equivalent, Fibroblasts, Flg, Human skin equivalent, HSE, Inv, iPSC, Keratinocytes, KGF, Lam,Layer, Lor, Maturation, Medium, Microenvironment, Organoid, NHDF, NHEK, Pharmacotoxicity,Proliferation, Regenerative medicine, SE, Signaling, TEM, Tissue engineering, Wound healing* Corresponding authors: [email protected]; [email protected]** These authors have contributed equally to this work and share senior authorship.This is an Open Access article distributed under the terms of the Creative Commons Attribution License h permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
2A. Idrees et al.: 4open 2021, 4, 1IntroductionHuman skin constructs (HSCs) are three-dimensional(3D) in vitro tissue-engineered human skin, which may ﬁndapplication in regenerative medicine and as in vitro modelsfor fundamental research and industrial uses, for examplefor cytotoxicity analysis, and testing new therapeuticapproaches. In advanced 3D in vitro systems, keratinocytesare able to develop well-ordered epidermis and basementmembrane features [1–3], to closely recapitulate the characteristics of native human skin (NHS). On the other side,ﬁbroblasts have an important role in enhancing thekeratinocytes resistance towards toxic compounds . Thissuggests that a single compartment skin model may not bepredictive for in vitro toxicological studies.As HSCs are physiologically more similar to the NHS,they offer advanced 3D testing systems as an alternativeto animals for drugs and cosmetics evaluation, irritancyand toxicity testing, wound healing studies, cancer research,skin infection studies, and research on other skin diseases[5–7]. Human and animal skin (e.g. mouse) have differentstructures, functionalities and responsiveness. Murine epidermis is quite thin composed of only three layers with ahigh turnover rate and present a cutaneous muscle layer,while human epidermis is thick, composed of six to tenlayers but lacks the muscle layer . Moreover, mouse skinis effectively able to regenerate after wounding, whilehuman skin damage may lead to hypertrophic scar (keloids)formation .Replacing low- to non-transferable in vivo animalexperiments and in vitro 3D animal skin models within vitro 3D skin models constructed from human cells is inline with the European Union regulations, 7th amendment(Dir. 2003/15/EC) of the “Cosmetics Directive” (76/768/EEC) which made obligatory to replace animal trials forcutaneous resorption with reliable in vitro tests by the year2009 , leading to the development of the 3Rs principle“Replacement, Reduction and Reﬁnement” .Extracellular matrix (ECM) is the mechanical supportand main component of 3D tissue microenvironment. TheECM also serves as a lead structure for diffusible molecules,including growth factors, which bind to ECM macromolecules and regulate inter- and intracellular communication via signalling pathways. It plays a key role inphysiological tissue homeostasis, growth and differentiation,but in pathological processes as well. Furthermore, themutual interactions between ECM and cells by mechanotransduction, result in speciﬁc cell surface receptor expression (e.g. integrins) , metabolic functions, cellproliferative potential, ECM production and release of keyregulators [13–20]. 2D cell cultures fail to reproduce thesefeatures and result in non-predictive outcomes mainly dueto forcing cells to adapt to an artiﬁcial ﬂat 2D surface .Cells grown in 3D environment may replicate the relevantcomplexity and dynamicity of the in vivo microenvironment.Up to now, studies on in vitro engineered humandermal-epidermal based skin models have been limited,while simple but poorly predictive epidermal models havebeen widely investigated . Although progresses havebeen made on differentiating mouse and human inducedpluripotent stem cells (iPSCs) in fully functional skin cells, and human skin organoids have been developed ,these models completely submerged in medium are not ableto recapitulate the physiological air-liquid interface environment or may still contain a heterogenous cell populationwith undifferentiated stem cells [24, 25].Advances in tissue engineering, cell transplantation, andgene therapy allow to obtain pure human cell cultures withhigh quality. However, the standardization of the procedures, especially for proliferative keratinocytes preparationas well as for their maintenance is a major obstacle. Additionally, the use of serum and other medium componentsis associated with batch-to-batch variations . Animalfree compounds and recombinant human growth factorscould represent a valuable alternative .In this study, 3D cell culture conditions (3D-CCs) andculture at air-liquid interface (ALI) were optimized todevelop full-thickness human skin equivalent (HSE) usinghuman primary cells. To achieve this aim, a gradual shifttowards the use of serum- and animal component-free cellculture media was performed to optimize HSE developmentto obtain thus dermal-epidermal constructs closely mimicking human skin.The HSE was based on a dermal compartment consisting of ﬁbroblasts embedded in collagen type I (Col. I) and amulti-layered, well differentiated epidermal compartment.Unlike epidermal or dermal skin models, full-thickness skinmodels comprising both dermal and epidermal compartments are advantageous. Firstly, ﬁbroblasts secrete growthfactors (e.g. keratinocyte growth factor, KGF) whichdirectly inﬂuence the growth and differentiation of keratinocytes [3, 28, 29]. Soluble factors secreted by dermalﬁbroblasts are diffused to the overlying epidermis, inﬂuencing the keratinocytes to induce the synthesis of basementmembrane proteins (i.e. laminin, collagen type IV, perlecanand nidogen) [1, 3, 30–32]. On the other hand, keratinocytesby secreting interleukin-1 (IL-1), also directly inﬂuence theproliferation of ﬁbroblasts . Moreover, the interactionbetween these two cell types is also highly important inthe formation of the basement membrane as a part ofdermal-epidermal junction (DEJ) [33, 34]. Thus, the reciprocal interaction works as a double-paracrine mechanismregulating each cell type [35, 36]. This cell-cell communication is mediated by hormones, growth factors, cytokinesand other signalling molecules that are secreted by a celland act on cells in its immediate vicinity and vice versa.The goal of this work was to develop an open-source andwell-reproducible 3D HSC as in vitro model of human fullskin, that can be helpful for preclinical testing, in compliance with the 3Rs principle.Materials and methodsCell source and maintenancePrimary cells including normal human dermal ﬁbroblasts (NHDF; CD90 positive) and normal human epidermalkeratinocytes (NHEK; Cytokeratin positive) were obtained
A. Idrees et al.: 4open 2021, 4, 1from PromoCell. The company guaranteed the followingrequested speciﬁcations: Same batch, single adult donor,chest region, and passage number P2 after thawing.NHDF and NHEK were maintained in CnT-Primeﬁbroblast medium (CnT-PR-F, CELLnTECH) and CnTPrime epithelial culture medium (CnT-PR, CELLnTECH)respectively under the physiological culture conditions(37 C, 5% CO2), and sub-cultured using DetachKit-PromoCell HEPES BSS ic acid buffered saline solution); 0.04% Trypsin/0.03% EDTA (ethylenediaminetetraacetic acid); andTNS (Trypsin Neutralizing Solution) containing 0.05%trypsin inhibitor from soybean/0.1% bovine serum albumin.Collagen type I (Col. I) from rat tail tendons, 5 mg/mLstock concentration was purchased from Ibidi.Different media were used to optimize the developmentof HSE: i) CnT-PR-F (CnT-Prime ﬁbroblast medium,CELLnTECH) named as Medium A; ii) CnT-PR (CnTPrime epithelial culture medium, CELLnTECH) namedas Medium B; iii) CnT-PR-3D (CnT-Prime-3D barriermedium, CELLnTECH) named as Medium C; iv) CnTPR-FTAL (CnT-Prime-full thickness airlift medium,CELLnTECH); v) KGM2 (Keratinocyte Growth Medium2, i.e. Basal medium supplemented with Bovine PituitaryExtract 0.004 mL/mL, human EGF 0.015 ng/mL, humanInsulin 5 lg/mL, Hydrocortisone 0.33 lg/mL, Epinephrine0.39 lg/mL, Transferrin 10 lg/mL, CaCl2 0.06 mM; PromoCell); vi) High calcium KGM2 (KGM2 1.2 mM CaCl2,Sigma); vii) FGM2 is a low serum media (2% v/v) that contains: 0.02 mL/mL foetal calf serum (FCS), 1 ng/mL basicﬁbroblasts growth factor (bFGF) (recombinant human),5 lg/mL Insulin (recombinant human) (PromoCell).Preparation of Human Skin Equivalents (HSEs)The 3D HSEs were generated by successively fabricating dermal and epidermal layers as summarized inFigure 1A.Preparation of Human Dermal Constructs (HDCs)Gelation of Col. I solution was performed in 10 media(M199, Sigma) supplemented with L-glutamine and sodiumbicarbonate (NaHCO3), resulting in a ﬁnal Col. I concentration of 1.5 mg/mL (containing a salt concentration of 1 inﬁnal mixture with pH of 7.2–7.4) (Tab. 1)HDCs were prepared by fabricating acellular (200 lL)and cell containing layers (400 lL) of Col. I matrix on polyester membrane of 12 well inserts (Corning 3460, 0.4 lm poresize, 12 mm diameter, 1.12 cm2 surface area) to obtain 5 mmthick HDCs. A thin layer of acellular collagen served as asubstrate for the cellular collagen, preventing the cellularcollagen from complete contraction from the insert membrane. Actively dividing mitotic cells (5 104 NHDF cellsper 12 mm diameter insert to obtain the human in vitro dermal construct) were embedded in Col. I solution and addedon the top of the previously deposited and gelled acellularCol. I layer (room temperature, 20 min). After gelation ofcellularized matrix (37 C for 30 min), fresh cell culturemedium (Medium A) was added and incubated for 5–7 days3to allow hydrogel remodelling by the embedded cells(Figs. 2A and 2B).Development of epidermis onto human dermalconstructsMedia was removed from HDC 20 min before NHEKseeding. NHEK were resuspended (400,000 cells for a surface area of dermal construct of 12 mm diameter) in a smallvolume of 50 lL KGM2 (3D-CC-I and II), CnT-PR (3DCC-III) or CnT-PR-FTAL (3D-CC-IV) in the center andplated onto the matrix for cultures epithelialization. Plateswere not moved for 15 min to allow keratinocytes to initiateadhesion and then placed at 37 C for 30–60 min withoutmedium for complete adhesion. Depending on the selectedculture conditions 3D-CC-I–IV, the plates were fed withthe appropriate media KGM2, CnT-PR (Medium B) orCnT-PR-FTAL as shown in Table 2. The Transwell insertswere placed in 12 well cell culture plate (Corning 3513),thus providing medium volumes of 0.3 mL inside and of 1.7 mL outside the insert. Medium was changed daily bydiscarding ﬁrst medium from outside the insert and thenfrom inside; fresh medium was then added initially insidethe insert and then outside. Cultures were maintained insubmerged conditions for 3–4 days to allow complete coverage to form a monolayer. In case of culture condition 3DCC-II KGM2 was replaced with CnT-PR-3D (Medium C)and for 3D-CC-III Medium B was replaced with MediumC (Tab. 2). For both culture conditions cultures were keptsubmerged overnight (15–16 h) to allow cells to form intercellular adhesion structures. Finally, the medium was aspirated from inside the insert to allow ALI culture for 20 days(Figs. 2C–2E). During ALI, medium was changed 3 times/week using a minimum volume of 1 mL.Different 3D cell culture conditions (namely 3D-CC-I,-II, -III, -IV) based on different types of commercially available media were tested to optimize HSE development. Thedetails of the 3D-CC are reported in Table 2. The test setup is described as timeline in Figure 1B.In this study, the same batch (single donor, chestregion) and passage number (P2 after thawing) of ﬁbroblasts and keratinocytes were used for optimizing the HSEdevelopment under different 3D culture conditions, thuseliminating the variability associated with batch-to-batchvariation.Morphological analysisHarvested HSE tissue was rinsed in PBS twice, ﬁxed in4% buffered paraformaldehyde (PFA, pH 6.8 – 7.0) for 4 h, dehydrated through increasing gradient series of ethanol (50%, 70%, 80%, 90%, 95%,100%, 100%, 100%; each for5 min), cleared in xylol (three times, each for 5 min), andinﬁltrated in parafﬁn using Leica TP1020 Semi-enclosedBenchtop Tissue Processor (over a total period of 12 h).The processed tissue was parafﬁn embedded at right orientation using Leica EG1160 Embedding Centre. The samplewas thinly sliced ( 5 lm) using Leica RM 2155 microtome.Sections were dryed at 37 C overnight. Morphological
4A. Idrees et al.: 4open 2021, 4, 1Fig. 1. (A) Schematic overview of methodology for the development of HSE. It starts with the embedding of human dermalﬁbroblasts in collagen I matrix to contract and remodel the gel to form dermal layer. Then the keratinocytes are seeded on the top toform a monolayer and the culture is raised to ALI under 3D cell culture conditions allowing the keratinocytes to differentiate and formepidermal layers. (B) Timeline for the development of HSE.Table 1. Guidelines for construction of collagen I (1.5 mg/mL) based dermal layer.ConstituentsCalculated vol. of each constituent (lL)Calculated vol. of each constituent (lL)Total vol. of acellular layer 200 lL per insertTotal vol. of cellular layer 400 lL per insert40 lL(10% of ﬁnal vol. of 400 lL results in 1 M199)1.36 lLFormula: M1·V1 M2·V2 (200 mM V1 0.68 400 lL) (This 1.36 lL vol. results in 0.68 mM Glutaminein ﬁnal 400 lL vol. of cellular layer)NHDF (PromoCell)20 lL(10% of ﬁnal vol. of 200 lL results in 1 M199)0.68 lLFormula: M1·V1 M2·V2 (200 mM V1 0.68 200 lL) (This 0.68 lL vol. results in 0.68mM Glutamine in ﬁnal200 lL vol. of acellular layer)113.32 lL(vol. calculated in the end)6 lL(This volume is 10% of the volume of collagen I, thatresults in a ﬁnal mixture pH of 7.2–7.4 – optimized)60 lL(This volume makes ﬁnal conc.of 1.5 mg/mL in ﬁnal volume)N/ATotal vol.200 lL10 M199 (Sigma)200 mM Glutamine(Biosciences)ddH2O7.5% of NaHCO3(Sigma)5 mg/mL Collagen I(Ibidi)analysis was performed on formalin-ﬁxed parafﬁnembedded sections through Haematoxylin and Eosin(H&E) staining following a standard protocol . TheH&E stained slides were examined with Olympus BX51light microscope. NHDF embedded in Col. I (Fig. 1A) were176.64 lL(vol. calculated in the end)12 lL(This is an optimized volume as 10% of the volume ofcollagen I, that results in a ﬁnal mixture pH of 7.2–7.4)120 lL(This volume makes ﬁnal conc. of 1.5 mg/mL in ﬁnalvolume)50 lL(This volume should contain5 104 NHDF per insert)400 lLvisualized by applying F-actin/nuclei staining. This wasperformed using Phalloidin/DAPI stain from Promokinefollowing a standard protocol . The ﬂuorescence microscopy and Z-stack imaging was performed using OlympusXM10 with cellSense Standard software.
A. Idrees et al.: 4open 2021, 4, 15Fig. 2. Macroscopic view of Human Dermal Construct (HDC) – upper panel: (A) View of experimental set-up of Human DermalConstruct on Day-01 with NHDFs are embedded in collagen I matrix and incubated to allow gel remodeling. (B) Macroscopic top viewof contracted collagen on Day-07 retaining its circular shape. Macroscopic view of HSE – lower panel: (C) Top view of experimentalset-up of HSE development during incubation at ALI showing the white epidermal layer over the dermal layer. (D) Side view of HSEcontaining insert at the time of tissue harvesting after 20 days at ALI, showing a thickness of 2 mm. (E) Top view of harvested HSEat the end of ALI period (20 days) retaining the size and diameter of initial mould (12 mm in diameter).Table 2. Experimental set-up details of different 3D cell culture conditions (3D-CCs).Step durationDescription of 3D culturecondition5–7 days3–4 days15–16 h10/15/20 days3D-CC-I3D-CC-II3D-CC-III3D-CC-IVHDC feedingFGM2FGM2Epidermal monolayerformation (onto HDC); feedingEpidermal monolayerformation (onto HDC); feedingHSC culture at ALI for10, 15, and/or 20 daysKGM2KGM2CnT-PR-F(Medium A)CnT-PR-FTAL–CnT-PR-3D(Medium C)CnT-PR-3D(Medium C)CnT-PR-F(Medium A)CnT-PR(Medium B)CnT-PR-3D(Medium C)CnT-PR-3D(Medium C)1.2 mM CaCl2containing KGM2–CnT-PR-FTALAbbreviations: CnT-PR-F named as Medium A; CnT-PR named as Medium B; CnT-PR-3D named as Medium C.Immunophenotypic analysisHSE was rinsed in phosphate buffered saline (PBS)twice, treated with 2M sucrose solution (Fisher Chemical)at 4 C for at least 1 h and then inﬁltrated in OCT (Optimal Cutting Temperature) embedding medium (VWR) for20–30 min at room temperature (RT). The tissue wasembedded in OCT by gradual freezing in liquid nitrogenvapor, stored at 80 C and cryo-sectioned ( 10 lm) usingMicrom HM 550 Cryostat.Immunophenotypic analysis of epidermal markers wasperformed on cryosections. Speciﬁcations on the primaryantibodies’ concentrations used in this study are describedin Table 3. Secondary antibodies used are the following:Alexa Fluor 488 Goat anti-Rabbit IgG (H L) (Abcam),Alexa Fluor 594 Donkey anti-Mouse IgG (H L) (Abcam),Alexa Fluor 488 Donkey anti-Rabbit IgG (H L)(ThermoFischer), Alexa Fluor 568 Goat anti-Mouse IgG(H L) (ThermoFischer). Negative controls wereperformed using only secondary antibodies. Cell nuclei were
6A. Idrees et al.: 4open 2021, 4, 1Table 3. Primary antibodies used for immunostaining of HSE tissue cryosections.Primary antibody details and sourceKi-67 (Rb, MA) – Cell signalLaminin 5 (Rb, PA) – AbcamKeratin 14 (Ms, MA) – AbcamKeratin 10 (Rb, MA) – AbcamFilaggrin (Rb, PA) – Abcamb-Tubulin (Ms, MA) – SigmaLoricrin (Rb, PA) – AbcamKeratin 16 (Rb, PA) – 001:2001:100 & 2 Ab as 1:200D2H10–LL002EP1607IHCY–EPR16774––PA polyclonal antibodies; MA monoclonal antibodies; Rb rabbit; Ms mouse; 2 Ab secondary antibodies.Table 4. Details of reagents required for Immunostaining.Reagent TypeReagent constitutionReagent Quantity (calculated)Wash bufferTBS 0.025% Triton X-100Permeabilization reagentFixativeAntibody diluent bufferBlocking Buffer0.2% Triton X-100 in PBS4% paraformaldehyde in PBSTBS 1% BSA10% normal serum 1% BSA TBSFor double staining: 5% normal serum of species inwhich ﬁrst secondary antibodies were raised 5%normal serum of species in which second type ofsecondary antibodies was raised 1% BSA TBSNote: Goat and donkey serum 60 mg/mL – JacksonImmuno Research1 TBS (10 TBS comprised of 500 mM trisbase 1.5 M NaCl HCl to set pH to 7.4); 1 PBS250 lL (0.25 mL) Triton X-100 in 1000 mLTBS20 lL (0.02 mL) Triton X-100 in 10 mL TBS10 mL of 16% PFA in 30 mL PBS0.1 g BSA in 10 mL TBS1mL normal serum 0.1 g BSA 9 mL TBSBuffers100 mL of 10 in 900 mL distilled deionizedH2OBSA is bovine serum albumin, TBS is tris-buffered saline, PBS is phosphate-buffered saline.counterstained with DAPI (40 ,6-diamidino-2-phenylindole)(1.5 lg/mL, VECTASHIELD, Vector labs). Immunohistochemical (IHC) analysis was performed in accordance withthe standard protocol from Abcam, with primary antibodies’ concentrations optimized for HSE (Tab. 3). Details ofreagents used in IHC staining are provided in Table 4.Cryosections were ﬁxed in methanol-free 4% PFA (ThermoScientiﬁc) for 10 min then rinsed in PBS (2 times for 5 min).Sections were then permeabilized in 0.2% Triton X-100 for10 min then rinsed in wash buffer (TBS, tris-buffered saline,0.025% Triton X-100). Sections were blocked in blockingbuffer (10% normal serum from the same secondaryantibody species, 1% BSA, Bovine Serum Albumin, inTBS) for 2 h at room temperature. Slides were drainedand treated with primary antibody diluted in antibody diluent buffer (1% BSA in TBS) and incubated in a humidiﬁedchamber overnight at 4 C. Fluorophore-conjugated secondary antibody diluted in antibody diluent buffer wereapplied and incubated for 1 h at room temperature, protected from light. Finally, sections were rinsed in TBS(3 times for 5 min) and mounted using VECTASHIELDmounting medium. The immunostained slides were examined with ﬂuorescent microscope (Olympus XM10 with cellSense Standard software).The percentage of Ki-67 positive dermal ﬁbroblasts andbasal keratinocytes was calculated from three IHC images(n 3 means three cryosectioned slices from a singleHSE, technical replicates and N 2, number of independent experiments) by dividing the Ki-67 positive nuclei(green) by total number of nuclei (blue). The equationapplied is as follows: [N(Ki-67 positive cell nuclei)/Ntotal(R(DAPI-stained cell nuclei, Ki-67 positive cells))] 100 % (Ki-67 positive cells).Electron microscopy analyses: SEM and TEMSamples were ﬁxed in 2.5% glutaraldehyde for 1 h,washed in PBS (3 times for 5 min), incubated with 1%osmium tetraoxide for 15 min, followed by PBS washing(3 times for 5 min). Then, samples were dehydrated in anincreasing gradient series of ethanol (30%-5 min, 50%5 min, 70%-overnight at 4 C, 96%-5 min for 3 times,100%-15 min for 3 times) and submerged in polypropyleneoxide (PO) for 15 min before inclusion in epoxy resin-basedembedding medium (Epoxy Embedding Medium kitcat.-no. 45359, Sigma-Aldrich) with PO (1:1) for 1 h .Thereafter, samples were embedded in the above mentionedmedium in the right orientation and incubated at 80 C for8 h. Tissue blocks were ultrasectioned ( 40 nm) using LeicaUltracut UCT Ultramicrotome, contrasted with Leicaultrastain 2 (3% lead citrate) and Leica ultrastain (0.5%uranyl acetate), and imaged with JEM-2100 TEM at anaccelerating voltage of 200 kV.
A. Idrees et al.: 4open 2021, 4, 1For scanning electron microscopy (SEM), samples wereﬁxed and dehydrated as described above. Then, sampleswere placed in BAL-TEC CPD 030 Critical Point Dryerand dryed with liquid CO2 by short consecutive immersions(3 times for 10 min). Then, samples were mounted ontoelectrically conductive, double sided adhesive carbon discs(Leit C tabs) and gold coated using Edwards S150B Sputter Coater under Argon gas (10 1 mbar) at voltage of15 mA for 6 min to form 40 nm thick gold (Au) coating.Coated samples were examined with SEM DSM 982 Gemini, Zeiss. Multiple SEM images were taken at random areasof different samples at 15 kV accelerating voltage. Thediameter of the collagen ﬁbers was estimated as an averagevalue from multiple TEM images.Wettability analysisStatic contact angle measurements were performed toinvestigate wettability of HSE by sessile-drop method,using custom-built contact angle goniometer instrumentunder ambient conditions. An amount of 5 lL MilliQ waterwas carefully placed on dry HSE prepared in two differentexperiments (N 2; Number of independent experiments);Multiple images were recorded (shown as an example inSupplementary Fig. 1S) and analyzed for 10 drops perHSE surface (N 2; n 10).Statistical analysisAll experiments, where not explicitly described otherwise, were performed independently in duplicate (N 2,number of independent experiments) and 3 technical replicates were made and examined per experiment (n 3).Standard deviations were given where appropriate andexpressed as mean SD (standard deviation). For statistical analysis, GraphPad Prism 5.00.288 (Inc., San Diego,CA, USA) was used to evaluate the signiﬁcance of the differences in experimental data. Signiﬁcance between groupswas considered for p 0.05.ResultsThe development of dermal-epidermal based HSEwas optimized to obtain well-differentiated epidermis ontoHDC. To this purpose, different 3D-CCs based on cellculture media and the parameter of ALI (Tab. 2) wereapplied showing signiﬁcant impact on tissue morphogenesis.Tissue morphogenesis of HSCs underdifferent 3D-CCsFigure 3 shows histological images of HSCs obtainedusing 3D-CC-I and 3D-CC-II, respectively. Two structurallydistinct compartments epidermis and dermis were present.Epidermis was slightly better organized in 3D-CC-II (Figs. 3,2A–2D, 2A0 –2D0 ) than in 3D-CC-I (Figs 3; 1A–1C,1A0 –1C0 ), which on the contrary displayed larger gapsbetween the layers. When comparing both culture7conditions, it is noticeable that the stratum corneum (SC)in the case of 3D-CC-II forms a clearer or more coherentlayer. Furthermore, organization of epidermal layers under3D-CC-II signiﬁcantly improved when air-lift (ALI) culturephase was extended to 15 days (Fig. 3, 2A0 –2D0 ).Furthermore, compared to 3D-CC-IV (based onCnT-FTAL, Fig. 4), 3D-CC-III (based on CnT-PR-3D(Medium C), Fig. 5) demonstrated an improved differentiated epidermal formation.Among the tested conditions, improved morphogenesisresults were obtained under 3D-CC-III and at ALI for20 days, resulting in constructs better mimicking epithelialdifferentiation found in healthy human skin. Hence, suchHSC was termed as human skin equivalent (HSE)(Figs. 5A00 –5D00 ). The epidermal part showed improved differentiated layers of keratinocytes namely stratum basale(SB), spinosum (SS), granulosum (SG), and corneum (SC)(Fig. 6A), more comparable with its in vivo counterpart(Fig. 6B).IHC analysis of differentiation markers on HSEIHC characterization of HSE (Figs. 7.1A and 7.1B)showed the presence of speciﬁc epidermal differentiation markers. Cytokeratin-14 (K14) was expressed byepithelial cells in the SB (Fig. 7.1A), while the suprabasallayers displayed the presence of Cytokeratin-10 (K10)(Fig. 7.1B). Cells in basal layer were found positive for Ki67 (highlighted by the arrows), demonstrating an activestate of proliferation (Fig. 7.2E). After counting, HSE displayed 10.5 0.8% (p-value 0.05) Ki-67 positive basalkeratinocytes. The terminal differentiation of epidermiswas demonstrated by the spotted expression (highlightedby arrows) of ﬁlaggrin (Flg) in the SG (Fig. 7.1C). Loricrin(Lor), a major protein component of corniﬁed envelope andmarker of terminally differentiated epidermal cells, alsoshowed spotted expression in SC (highlighted by arrows inFig. 7.2F). Involucrin (Inv) was displayed in upper spinous,sub-corneal, and corneal layers (Fig. 7.2G). Laminin 5(Lam 5) was expressed at DEJ as a thin continuous line(Fig. 7.1D) suggesting stable epidermal-dermal interaction.Beta-tubulin as a component of the cytoskeletal microtubule
dermal-epidermal junction (DEJ) [33, 34]. Thus, the recip-rocal interaction works as a double-paracrine mechanism regulating each cell type [35, 36]. This cell-cell communica-tion is mediated by hormones, growth factors, cytokines and other signalling molecules that are secreted by a cell a