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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 [37]. 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 [47]. 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 [38].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