Bioengineering the microanatomy of human skin

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  Bioengineering the microanatomy of human skin Mathilde Roger, 1, * Nicola Fullard, 1, * Lydia Costello, 1, * Steven Bradbury, 1 Ewa Markiewicz, 1 Steven O’Reilly, 2 Nicole Darling, 1 Pamela Ritchie, 1 Arto M € a € att € a, 1 Iakowos Karakesisoglou, 1 Glyn Nelson, 3 Thomas von Zglinicki, 3 Teresa Dicolandrea, 4 Robert Isfort, 4 Charles Bascom 4 andStefan Przyborski 1,5 1 Department of Biosciences, Durham University, Durham, UK  2 Department of Health and Life Sciences, Northumbria University, Newcastle, UK  3 Institute for Ageing and Health, University of Newcastle, Newcastle, UK  4 Mason Business Centre, Procter & Gamble, Mason, Cincinnati, OH, USA 5 Reprocell Europe, Sedgefield, UK  Abstract Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testinginterventions, and reducing the use of animals. Critical to the use of such technology is the ability to producetissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell-based skinsystems lack thorough characterisation, are not representative of human skin, and can show variation. In thisstudy, we have developed a novel full thickness model of human skin comprised of epidermal and dermalcompartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts thatsecrete their own extracellular matrix proteins, which avoids the use of animal-derived materials. The dermalconstruct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into astratified keratinised epithelium. In-depth morphological analyses of the model demonstrated very closesimilarities with native human skin. Extensive immunostaining and electron microscopy analysis revealedultrastructural details such as keratohyalin granules and lamellar bodies within the  stratum granulosum ,specialised junctional complexes, and the presence of a basal lamina. These features reflect the functionalcharacteristics and barrier properties of the skin equivalent. Robustness and reproducibility of  in vitro  modelsare important attributes in experimental practice, and we demonstrate the consistency of the skin constructbetween different users. In summary, a new model of full thickness human skin has been developed thatpossesses microanatomical features reminiscent of native tissue. This skin model platform will be of significantinterest to scientists researching the structure and function of human skin. Key words:  barrier function; dermis; epidermis; human; methodology; reproducible; skin equivalent; tissueengineering. Introduction The skin is the largest organ of the human body, account-ing for approximately 16% of the total body weight. It isthe primary interface between the internal and externalenvironments which acts as a barrier to protect the bodyfrom a range of environmental stressors and maintainhomeostasis by preventing unregulated water and elec-trolyte loss. The anatomy of human skin is well charac-terised, and there is a clear relationship between itsstructure and function. It is therefore essential to considerthe structural aspects of skin when developing tissuemimetics to study skin health and disease, to ensure thatthe  in vitro  model accurately recapitulates the anatomicalfeatures of its native counterpart.The skin is a complex organ with two distinct structuralupper layers: the epidermis and the dermis. The epidermis isa stratified keratinised epithelium predominantly made upof keratinocytes, which is subdivided into four layers: basal,spinous, granular, and cornified. The basal layer rests upona basement membrane at the dermoepidermal junction. Itconsists of mitotically active columnar cells which prolifer-ate, migrate superficially, and sequentially differentiate toform the stratified epidermis. The basal layer is charac-terised by the expression of keratin 14, and the daughterkeratinocytes undergo a characteristic basal-to-suprabasal CorrespondenceStefan Przyborski, Department of Biosciences, Durham University,Durham, DH1 3LE, UK. E:  *Joint first authors.Accepted for publication  7 January 2019 ©   2019 The Authors.  Journal of Anatomy   published by John Wiley & Sons Ltd on behalf of Anatomical Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use,distribution and reproduction in any medium, provided the srcinal work is properly cited.  J. Anat.  (2019) doi: 10.1111/joa.12942 Journal of Anatomy  switch from keratin 5/14 to keratin 1/10 as they move upthe strata. As these cells move towards the surface and dif-ferentiate into the spinous layer, they lose their ability todivide, become larger, and establish robust intercellularconnections. Keratinocytes elongate and flatten to formthe granular layer, which is characterised by the presence ofintracellular keratohyalin granules and lamellar bodies(Odland, 1960). Keratohyalin granules contain proteins keyto the formation of the cornified envelope such as profilag-grin and loricrin (Yoneda et al. 1992). Keratinocytes are ter-minally differentiated into corneocytes, which make up the  stratum corneum . During this transition, cells lose theirnuclei and major organelles, lipids are released into theintercellular space and the cornified envelope replaces thecell membrane. The barrier function of the skin is mainlyattributed to the  stratum corneum , which is a 10- to 20- l m-thick layer composed of terminally differentiated, flattenedcorneocytes separated by layers of densely packed lipids(Menon et al. 2012).The dermis provides crucial support to the epidermis.Unlike the epidermis, vascular and lymphatic systems per-vade the dermis alongside appendages such as hair folli-cles, nerve endings, and secretory glands. The primary celltype in the dermis is the fibroblast. They produce extracel-lular matrix proteins (ECM) (e.g. collagen, fibronectin, andelastin), which contribute to the main substance of the der-mis and are responsible for skin elasticity and tensilestrength. The dermoepidermal junction separates the der-mis and epidermis, and it facilitates the regulatedexchange of substances and the polarity of the basal ker-atinocytes (Muroyama & Lechler, 2012). Collagen IV andintegrin  a 6 are critical components of the basement mem-brane, which also contribute to the mechanical integrity ofthe skin.Human skin equivalent models are important tools foracademic research, clinical purposes, and industrial applica-tions. The need for physiologically relevant models is imper-ative due to the recent prohibition on the use of animalsfor testing active compounds for cosmetics (Cosmetics regu-lation EC No 1223/2009). However, due to the multicellular,multi-layered complexity of human skin, it is highly chal-lenging to build such tissue models reproducibly and in aconsistent manner. Engineered models are incrementallyadvancing in emulating the anatomy of skin by focusing onthe co-culture of the main cell types (keratinocytes, fibrob-lasts and, less frequently, melanocytes). To achieve epider-mal differentiation,  in vitro  models are commonly raised tothe air  –  liquid interface, and additives, such as growth fac-tors and calcium, promote differentiation and stratificationto recreate an  in vivo -like stratified keratinised epidermis(Prunieras et al. 1983; Bikle et al. 2012).Epidermal models are most often generated on a poly-carbonate membrane; however, due to the presence ofkeratinocytes alone, they lack the key interactions withdermal cells (Valyi-Nagy et al. 1990).  In vivo , the epidermisis bound tightly to the dermal component via the base-ment membrane, which is composed of ECM proteinssecreted as a result of interactions between keratinocytesand fibroblasts (Jahoda et al. 2001). To overcome thisissue, epidermal models often require the use of exoge-nous ECM coating to enable keratinocyte adhesion to theinert membrane.More complex full thickness skin models include both thedermal and epidermal components. Pioneering work in thedevelopment of skin constructs initially involved seedingkeratinocytes on to decellularised pig skin (Freeman et al.1976) or human de-epidermised dermis (Prunieras et al.1983). More recently, the development of three-dimen-sional (3D) technologies has rapidly advanced skin bioengi-neering within the laboratory. Full thickness models ofmammalian skin have been created using hydrogel-basedtechnologies (usually collagen-based) into which fibroblastsare integrated (Gangatirkar et al. 2007; Carlson et al. 2008;El Ghalbzouri et al. 2009). Although these systems providegood support for keratinocyte differentiation and stratifica-tion, there are several disadvantages, including the use ofanimal-derived collagen, contraction of the collagen gel,and use of exogenous collagen matrix that can introducebatch-to-batch variability (El Ghalbzouri et al. 2005). More-over, there is evidence to suggest that the particular com-position and arrangement of the ECM can influence thephenotype of the dermal fibroblasts and thus their abilityto support the epidermis (Maas-Szabowski et al. 2003; Sor-rell & Caplan, 2004).An alternative strategy is to create an environment thatenables the fibroblasts to generate their own ECM matrix.To achieve this, models have been generated by layeringsheets of fibroblasts to mimic the dermal compartment,before the addition of keratinocytes. This approach resultedin a stratified epidermis but lacked a true 3D structure, andanalysis by transmission electron microscopy showed incom-plete basement membrane formation (Lee et al. 2009).More recently, we have grown primary human dermalfibroblasts in an inert polystyrene scaffold, which enablesthe deposition of ECM components to support overlyingepithelial cells and epidermal stratification (Hill et al. 2015).However, differences in cells between donors introducedsignificant structural variability in the consistency and repro-ducibility of the models.Although numerous approaches exist to generate in vitro  models of human skin, a common drawback ofboth epidermal and full thickness systems is the complex-ity of the methods used to generate them. Protocols usu-ally rely on complex in-house media recipes that containmultiple additives such as hydrocortisone, insulin, transfer-rin, and epidermal growth factor (Bertolero et al. 1984).The addition of such ingredients and the use of high con-centrations of serum decrease the reproducibility betweenlaboratories and make these complex models even moredifficult to replicate (Faller & Bracher, 2002; Ng & Ikeda, ©   2019 The Authors.  Journal of Anatomy   published by John Wiley & Sons Ltd on behalf of Anatomical Society.Bioengineering the anatomy of human skin, M. Roger et al. 2  2011). Moreover, the nature of cells used in skin equiva-lents can compromise the ability to generate skin modelsreproducibly. Primary cells, especially keratinocytes, arenot all able to differentiate and stratify  in vitro  and cancause unpredictability (Stark et al. 1999; Eves et al. 2000).Attempts to use cell lines, such as HaCaT cells, haveshown disorganised morphology (Boelsma et al. 1999;Maas-Szabowski et al. 2003; Stark et al. 2004). Recently,Reijnders et al. successfully developed a full thicknessmodel using TERT-immortalised keratinocytes and fibrob-lasts (Reijnders et al. 2015). However, independent of thecells used, the ability to differentiate, stratify, and accu-rately reproduce the microanatomy of the human skinhas to be confirmed. Skin models using commerciallyavailable cells, more clearly defined media, and additiveswould help to reduce skin structure variability signifi-cantly and enhance intra- and inter-laboratory repro-ducibility.Many researchers rely on commercially available systemssuch as EpiSkin  , EpiDerm TM , SkinEthic  , and the LabCyteEPI-MODEL 24 for epidermal models (El Ghalbzouri et al.2008; Mathes et al. 2014), and Phenion Full Thickness,Stratatest and Epiderm-FT for full thickness models (Scha-fer-Korting et al. 2008; Ackermann et al. 2010; Rasmussenet al. 2010). A range of assays can be performed usingthese models, which include wound healing, skin hydra-tion, drug delivery and phototoxicity. However, the meth-ods used to generate these skin equivalents are nottransparent, the models arrive pre-made, and they lackthe flexibility to be tailored for specific downstream appli-cations. Furthermore, although basic characterisation ofthese models has been reported (Ponec et al. 2001, 2002;Ponec, 2002; Botham, 2004), it is incomplete and there islittle in-depth analysis of the microanatomy of these con-structs, particularly in comparison with the structure ofhuman skin.In this study, we report the development of novel strate-gies to generate robust and reproducible models of thehuman epidermis and human full thickness skin that closelymimic aspects of the architecture of skin tissue. Theapproaches incorporate the use of batches of commerciallyavailable primary cells isolated from single donors anddefined low serum media, which requires only three addi-tives for epidermal stratification. Neonatal cells were chosenfor this study due to their proliferative capacity andreduced variability, as they are derived from donors of asimilar age. They enable the generation of a dermal modelthat does not require the addition of exogenous collagenand relies on the endogenous deposition of ECM fromhuman dermal fibroblasts. The dermal equivalent supportsthe formation of a highly differentiated epidermis and thecreation of a protective barrier. In-depth analysis of themicroanatomy of these models demonstrates their similarityto human skin and their use as valuable tools to study thestructure and function of human skin. Materials and methods Primary cells and cell maintenance Human neonatal epidermal keratinocytes (HEKn, Thermo Fisher Sci-entific, Loughborough, UK) were maintained in KeratinocyteGrowth Medium composed of EpiLife  medium, (Thermo Fisher Sci-entific), supplemented with human keratinocyte growth supple-ment (HKGS, Thermo Fisher Scientific) and 10  l g mL  1 gentamicinand 0.25  l g mL  1 amphotericin B (Thermo Fisher Scientific), at37  ° C in a 5% CO 2  humidified incubator following the supplier’sinstructions.Human neonatal dermal fibroblasts (HDFn, Thermo Fisher Scien-tific) were maintained in Dermal Fibroblast Growth Medium com-prised of Medium 106 (Thermo Fisher Scientific), supplementedwith low serum growth supplement (LSGS, Thermo Fisher Scientific),10  l g mL  1 gentamicin, and 0.25  l g mL  1 amphotericin B (ThermoFisher Scientific), at 37  ° C in a 5% CO 2  humidified incubator follow-ing the supplier’s instructions. Skin equivalent generation Epidermal models were generated in Millicell  cell culture inserts(Merck Millipore, Beeston, UK) by coating with human collagen Idiluted 1 : 100 (Thermo Fisher Scientific). HEKn were trypsinised,centrifuged, and 5  9  10 5 cells were re-suspended in KeratinocyteGrowth Medium supplemented with 5 ng mL  1 keratinocytegrowth factor (KGF; Thermo Fisher Scientific), 140  l M  CaCl 2 (Sigma-Aldrich, Dorset, UK), and 50  l g mL  1 ascorbic acid (Sigma-Aldrich). Cells were then seeded on the collagen-coated insertand incubated at 37  ° C in a humidified 5% CO 2  incubator. After2 days, the models were raised to the air  –  liquid interface andsupplemented with 1.64 m M  CaCl 2  and maintained up to a fur-ther 28 days.Dermal models were generated by seeding HDFn (5  9  10 5 cells)onto inert porous polystyrene membranes (12-well Alvetex  scaf-fold inserts, Reprocell) and incubating at 37  ° C in a 5% CO 2  humidi-fied incubator in Dermal Fibroblast Growth Medium supplementedwith 5 ng mL  1 transforming growth factor (TGF b )1 (Thermo FisherScientific) and 100  l g mL  1 ascorbic acid. Dermal equivalents weremaintained for up to 35 days.Full thickness human skin models were generated by seeding1.3  9  10 6 HEKn cells onto dermal equivalents in KeratinocyteGrowth Medium supplemented with 10 ng mL  1 KGF, 140  l M  CaCl 2 and 100  l g mL  1 ascorbic acid, and incubating at 37  ° C in a 5%CO 2  incubator. After 48 h, the models were raised to the air  –  liquidinterface and cultured in Keratinocyte Growth Medium supple-mented with 10 ng mL  1 KGF, 1.64 m M  CaCl 2  and 100  l g mL  1 ascorbic acid and maintained up to a further 28 days. Human skin samples Skin biopsies from young adult Caucasian women were collected byProcter and Gamble USA, under an IRB-approved clinical protocolin compliance with local laws and regulations. Paraffin embedding Skin equivalents and human skin samples were fixed in 10% forma-lin (Sigma-Aldrich), gradually dehydrated in ethanol (30  –  100%) and ©   2019 The Authors.  Journal of Anatomy   published by John Wiley & Sons Ltd on behalf of Anatomical Society.Bioengineering the anatomy of human skin, M. Roger et al.  3  Histoclear (Thermo Fisher Scientific), and embedded in paraffin(Thermo Fisher Scientific) using plastic moulds (CellPath, Newton,UK) to allow for transverse sectioning. Using a microtome (LEICARM2125RT), 5- l m sections were transferred to charged superfrostmicroscope slides (Thermo Fisher Scientific). Histological analysis For haematoxylin & eosin (H&E) staining, sections were deparaf-finised in Histoclear and gradually rehydrated in ethanol. Slideswere incubated in Mayer’s haematoxylin (Sigma-Aldrich) for 5 minand rinsed in distilled water for 30 s before being incubated withalkaline alcohol for 30 s to ensure the nuclei appear blue. Sampleswere dehydrated, before being incubated with eosin (Sigma-Aldrich) for 30 s, and then further dehydrated prior to mountingwith DPX (Thermo Fisher Scientific) ready for microscopy using aLeica ICC50 high definition camera mounted onto a BrightfieldLeica microscope. Immunostaining For immunostaining, sections were deparaffinised in Histoclearand gradually rehydrated in ethanol. Antigen retrieval wasachieved by incubating samples in a 95  ° C water bath for20 min with citrate buffer (Sigma-Aldrich). Samples were thenincubated and permeabilised for 1 h in blocking buffer: 20%neonatal calf serum (NCS, Sigma-Aldrich) in 0.4% Triton X-100(Sigma-Aldrich) in phosphate-buffered saline (PBS). Primary anti-bodies diluted in blocking buffer (Table S1) were incubated withthe samples overnight at 4  ° C, followed by three washes in PBS.Samples were then incubated for 1 h at room temperature withthe relevant secondary antibody (donkey anti-rabbit Alexa Flu-or  488 or 594 or donkey anti-mouse Alexa Fluor  488 or 594,Thermo Fisher Scientific). After being washed three times in PBSsections were mounted using Vectashield/DAPI Hardset (VectorLaboratories, Peterborough, UK). The fluorescent images werecaptured using Zeiss 880 with Airyscan confocal microscope withZ EN  software. Collagen assay Total collagen was quantified using the QuickZyme kit (QuickZymeBiosciences, Leiden, The Netherlands) following the supplier’sinstructions. Briefly, snap-frozen dermal equivalents grown for 7  –  35 days and standards were hydrolysed in 6  M  HCl 3 mg  l L  1 wetweight (Thermo Fisher) for 20 h at 95  ° C, centrifuged for 10 min at13 000  g , and the supernatant collected. Samples were diluted inwater (for a final concentration of 4  M  HCl). All samples were incu-bated for 60 min at 60  ° C with detection reagent before absor-bance was read at 570 nm using a Biotek plate reader (Biotek,Swindon, UK). Total collagen values were calculated using the stan-dard and expressed in  l g mL  1 . Electron microscopy For transmission electron microscopy (TEM), samples were fixed inKarnovsky’s Fixative [8% paraformaldehyde, 25% glutaraldehyde(Agar Scientific, Stansted, UK), 0.2  M  cacodylate buffer (pH 7.4)(Agar Scientific) and distilled water] for 1 h at room temperaturethen washed three times for 5 min in 0.1  M  cacodylate Buffer (pH7.6) (Agar Scientific). Samples were further fixed in 1% osmiumtetroxide (Agar Scientific) for 1 h and then washed in 0.1  M cacodylate buffer (pH 7.6), prior to sequential dehydration in 50,70, 95, and 100% ethanol. Samples were then embedded in resinas follows: the samples were immersed in an intermediate solutionconsisting of a 50 : 50 mix of 100% alcohol and propylene oxide(Agar Scientific) for 15 min, moved to propylene oxide for 15 min,placed in a fresh 50 : 50 Agar 100 Epon resin : propylene oxide(Agar Scientific) mix for 15 min, and finally the samples wereplaced in Epon resin three times for 1 h each. The Agar 100 Eponresin was composed of 24 g Agar 100 Epon Resin, 9 g dodecenyl-succinic anhydride (DDSA), 15 g methyl nadic anhydride (MNA)and 1.4 g benzyldimethylamine (BDMA) (Agar Scientific). Thenspecimens were placed into rubber moulds with fresh Agar 100Epon resin, which was left to polymerise for 24 h at 60  ° C. Ultra-thin sections were cut using a diamond knife (Agar Scientific) on aReichert Ultracut S Ultramicrotome (Leica) and transferred to 200-mesh copper formvar-coated grids (Agar Scientific). Sections werethen stained with 1% uranyl acetate (BDH) in 70% ethanol,washed in water, and then stained with Reynolds’ Lead Citrate(BDH) for visualisation. Ultra-thin sections were imaged on aH7600 TEM (Hitachi).For scanning electron microscopy (SEM), samples were criticallydried (BAL-TEC CPD 030) (Leica) and coated with 5 nm platinum, ina Cressington Coating System 328 (Cressington). Sample visualisa-tion was performed using a S5200 scanning electron microscope(Hitachi). Barrier resistance assay The integrity of the epidermal barrier was assessed by challengingthe surface of the epithelium with detergent, followed by assess-ment of cell viability and the ability of a fluorescent dye to pene-trate the tissue model. Briefly, epidermal models grown for10 days at the air  –  liquid interface were transferred to 6-well platescontaining 0.9 mL of medium. To determine the concentration atwhich a marker chemical reduces the viability of the tissues by50% (IC 50 ) after a fixed expoure time, a range of concentrations ofsodium dodecyl sulphate (SDS; 0-4 mg mL  1 in water) were topi-cally applied (70  l L) for 18 h. After exposure, samples werewashed twice in PBS, dried carefully, and transferred to a 24-well Fig. 1  Formation and characterisation of epidermal construct. (A) Histological assessment of epidermal structure over time shown by representa-tive H&E images of epidermal models cultured for up to 28 days at the air  –  liquid interface. Scale bars: 100  l m. (B) Representative immunofluores-cence micrographs of the epidermal construct, cultured for 14 days at the air  –  liquid interface. Data show protein expression patterns for keybiomarkers in the epidermis. Scale bars: 50  l m. (C) Representative SEM and TEM micrographs of the epidermal model cultured for 14 days at theair  –  liquid interface. (C,a) Cross-section SEM micrographs, showing multiple cell layers with the  stratum corneum  demonstrating less adherencethan the layers below (white arrows). Scale bars: 25  l m. (C,b-d) TEM micrographs showing the Millicell   membrane (mem), hemidesmosome-likestructures (hm), desmosomes (ds), and keratin fibres (kf). (C,c) Electron-dense desmosome between cells at higher magnification. (C,d) Electron-dense junctional complex between cells and the underlying Millicell   membrane. Scale bars: 2  l m (C,a,b), 500 nm (C,c,d). ©   2019 The Authors.  Journal of Anatomy   published by John Wiley & Sons Ltd on behalf of Anatomical Society.Bioengineering the anatomy of human skin, M. Roger et al. 4
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