REGULAR ARTICLE

Three-dimensional culture and identification of human eccrinesweat glands in matrigel basement membrane matrix

Haihong Li & Lu Chen & Mingjun Zhang & Shijie Tang &

Xiaobing Fu

Received: 29 March 2013 /Accepted: 6 August 2013 /Published online: 31 August 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Interactions between the extracellular matrix(ECM) and epithelial cells are necessary for the proper orga-nization and function of the epithelium. In the present study,we show that human eccrine sweat gland epithelial cellscultured in matrigel, a representation of ECM components,constitute a good model for studying three-dimensional re-construction, wound repair and regeneration and differentia-tion of the human eccrine sweat gland. In matrigel, epithelialcells from the human eccrine sweat gland form tubular-likestructures and then the tubular-like structures coil into sphere-like shapes that structurally resemble human eccrine sweatglands in vivo. One sphere-like shape can be linked to anothersphere-like shape or to a cell monolayer via tubular-likestructures. Hematoxylin and eosin staining has revealed that thetubular-like structures have a single layer or stratified epithelialcells located peripherally and a lumen at the center, similar

to the secretory part or duct part, respectively, of the eccrinesweat gland in sections of skin tissue. Immunohistochemicalanalysis of the cultures has demonstrated that the cells expressCK7, CK19, epithelial membrane antigen and actin. Thus,matrigel promotes the organization and differentiation of epithe-lial cells from the human eccrine sweat gland into eccrine sweatgland tissues.

Keywords Eccrine sweat gland . Three-dimensional culture .

Matrigel . Tubular-like structure . Sphere-like structure .

Human

Introduction

The human body has 3×106 to 4×106 sweat glands, two typesof which are generally recognized, namely, the eccrine sweatgland and apocrine sweat gland. In the human being, theeccrine sweat gland is distributed all over the body surface,except for the lips, external ear canal, clitoris, labia minora,glans penis and nail bed (Shibasaki et al. 2006). The eccrinesweat gland is a single tubular structure, the deep part of whichis coiled into a ball; the gland traverses the dermis and epi-dermis and opens onto the surface of the skin (Shibasaki et al.2006). The main functions of the eccrine sweat gland are tocontrol the body temperature by the evaporation of sweat,with about 25 % of heat being eliminated in this way underbasal conditions and to protect the body against harmfulbacteria and other viruses (Cai et al. 2011; Grice and Segre2011). Therefore, the eccrine sweat gland is essential for thecontrol of body temperature for human beings. Patients withanhidrotic ectodermal dysplasia who congenitally lack eccrinesweat glands and those having experienced severe burns fol-lowing which most of the eccrine sweat glands have been lostwill suffer from increasing body temperature while exercisingor being in a warm environment (Zonana et al. 2000; Li et al.2006; Sheng et al. 2009).

This work was supported in part by the National Natural ScienceFoundation of China (81071551, 30500194), the Advanced Talent Itemof Colleges and Universities of Guangdong Province and the NationalBasic Science and Development Program (973 Program,2012CB518105).

The authors declare no competing financial, personal, or otherrelationships with other individuals or organizations.

H. Li (*) : L. Chen :M. Zhang : S. TangBurn and Plastic Surgery, The Second Affiliated Hospital, ShantouUniversity Medical College, North DongXia Road, Shantou,Guangdong Province 515041, People’s Republic of Chinae-mail: lihaihong1051@126.com

H. LiResearch Center for Translational Medicine, Shantou UniversityMedical College, North DongXia Road, Shantou, GuangdongProvince 515041, People’s Republic of China

X. Fu (*)Burns Institute, The First Affiliated Hospital, Chinese PLA GeneralHospital, Trauma Center of Postgraduate Medical School, 51 FuCheng Road, Beijing 100037, People’s Republic of Chinae-mail: fuxiaobing@vip.sina.com

Cell Tissue Res (2013) 354:897–902DOI 10.1007/s00441-013-1718-3

The development of tissue engineering has opened up anew path for the repair of large skin lesions and so thesurvival rate of severe burn and critically severe burn victimsis above 90 % in many countries (Fu et al. 2005). However,burn survivors often have difficulty in hot and humid condi-tions, because neither the commercially available products northe products currently described in experimental studies areable to substitute fully for natural living skin, the commonproblem being that none of them can rebuild hair follicles,sebaceous glands and sweat glands; this greatly affects thequality of life of such patients (Fu et al. 2005; Sheng et al.2009). Thus, the reconstruction of skin that possesses not onlythe epidermal and dermal parts but also skin appendages isimportant.

In vivo, cells exist in an organized three-dimensional (3D)structure and so cells cultivated in monolayers (2D) do notrecapitulate the physiological architecture of those in vivo. Inrecent years, with the development of extracellular matrices,the 3D reconstruction of the eccrine sweat gland has becomepossible (Kleinman and Martin 2005). In this study, humaneccrine sweat gland cells were harvested and cultured instandard monolayers and 3D matrices and their morphologywas recorded, histologically and immunohistochemically.

Materials and methods

Primary culture of eccrine sweat gland cells in monolayerculture

Non-cauterized and full-thickness skin specimens used for theisolation of eccrine sweat glands were obtained from individ-uals undergoing plastic surgery in the Burn and PlasticSurgery of the Second Affiliated Hospital of ShantouUniversity Medical College. The average age was about 20±2 years old and the regions of the specimens includedfingers, abdomens, arms, and legs. Ethical permission wasgranted by the ethics Committee of Shantou UniversityMedical College (Shantou, China) and informed consentwas obtained from patients or their guardians. Subcutaneousfat of the skin was removed and then the skin was rinsed withHank’s balanced salt solution (Gibco, USA) supplementedwith 100 IU/ml penicillin and 100 μg/ml streptomycin.Finally, the skin was minced as finely as possible with sharpscissors in a 60-mm plastic culture dish and then incubated in2 mg/ml collagenase type II (Gibco, USA) in DMEM/F12(1:1) medium containing 100 IU/ml penicillin, 100 μg/mlstreptomycin and 5 % fetal bovine serum at 37 °C in ahumidified atmosphere of 5 % CO2/95 % air in an incubatorfor 5–6 h. Following enzymic treatment, the eccrine sweatglands were released from peripheral tissues and at least threeseparated sweat glands were picked by using a 100-μl pipettorand transferred from one plastic culture dish to another under a

clean and infrared-sterilized inverted phase contrast micro-scope (Leica, Germany). The picked sweat glands were cul-tured in defined keratinocyte serum free medium (KSFM;Gibco) supplemented with 5 ng/ml recombinant human epi-dermal growth factor (EGF), 25 mg/ml bovine pituitary ex-tract (BPE), 100 U/ml penicillin and 100 μg/ml streptomycinat 37 °C in a humidified atmosphere of 5%CO2/95% air in anincubator. The medium was subsequently changed every 2 or3 days. When the cells reached more than 70 % confluence,the cells were harvested for 3D culture.

Three-dimensional culture of eccrine sweat gland cellsin matrigel

Aliquots of cells (5×104 cells) in KSFM (0.3 ml) were addedto 0.3 ml cold matrigel basement membrane mtrix (BDBiosciences, USA), suspended by using cooled pipettes andthen added to the wells of a 12-well Costar culture plate(Corning, USA). The plates were incubated at 37 °C for30 min to allow gel formation. When the gel had formed,defined KSFM could be added. The medium was subsequent-ly changed every other day. The 3D cultures were observedunder an inverted microscope.

Hematoxylin and eosin staining and immunohistochemicalanalysis of 3D cultures

After the sweat gland epithelial cells had been cultured inmatrigel for 12 days, the 3D cultures were embedded inoptimum cutting temperature compound (OCT) and frozensections (20 μm thick) were cut.

For hematoxylin and eosin (HE) staining, the sections werestained with HE, mounted in resin and observed under amicroscope.

For immunohistochemical analysis, the sections werestained with a two-step immunostaining kit (Zymed, USA).First, sections were treated with 3 % H2O2 to block endoge-nous peroxidase, followed by antigen retrieval according tothe requirements of each antigen. For antibodies against CK7and CK19, sections were heated to 95 °C in a citric acid buffer(pH 6.0) for 15 min and slowly cooled to room temperature;for antibodies against epithelial membrane antigen (EMA)and actin, sections needed no treatment. Subsequently, thesections were incubated at 4 °C overnight with the respectivemonoclonal mouse anti-human antibodies (NeoMarker, USA)at a dilution of 1:100, followed by treatment with Cy3-conjugated goat anti-mouse IgG (Beyotime, China) for30 min at 4 °C at a dilution of 1:500. Finally, sections werecounterstained with 4,6-diamidino-2-phenylindole (DAPI) tolabel the nucleus, mounted in anti-fade polyvinylpyrrolidonemounting medium (Beyotime, China) and observed under afluorescence microscope. Sections from which the primaryantibodies had been omitted were used as negative controls.

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Results

Primary 2D culture of eccrine sweat gland cells

Skin samples could be stored at 4 °C in DMEM supplementedwith 10 % fetal bovine serum (FBS) for 3 to 4 days. Whenskin samples were digested with collagenase type II for 4–5 h,most eccrine sweat glands were released from peripheraltissues (Fig. 1a). However, if the digestion timewas prolongedto 7 h or longer, the released eccrine sweat glands were easierto dissociate. By 24 h after seeding, the cultured tissues hadattached to the culture dish. The efficiency of outgrowth was60–80 %. The intervals from attachment to outgrowth of theexplants were variable ranging from 1 day to 7 days or evenslightly longer but most within 3–5 days. Epithelial cellsgrowing out from the explants were tightly connected andpresented their typical “cobblestone” morphology (Fig. 1b,c). As a cell monolayer continued to grow larger, many celllayers began to form over areas of previous growth.

Culture of eccrine sweat gland cells in matrigel

The mixture of cells and matrigel formed a clear gel whenwarmed to 37 °C within 30 min. After seeding, some of thecells grew in the matrigel and some under the matrigel, name-ly, they adhered to the plastic substrate. Cells cultured inmatrigel and under matrigel grew differently. During the cul-turing process, each cell (Fig. 2a) divided into 2–4 cells ondays 2 and 3 (Fig. 2b–d) and subsequently these divided cellsformed small cell clusters on days 4 and 5 (Fig. 2e, f) and largecell clusters on days 9 and 10 (Fig. 2g) when the cells grewwithin the matrigel. The cell clusters formed tubular-likestructures (Fig. 2e) and then the tubular-like structures coiledinto sphere-like shapes (Fig. 2f, g). However, when the cellsgrew under the matrigel, they formed a standard monolayerrather than the sphere-like shape (Fig. 2h). A sphere-like shapeoften linked itself to another sphere-like shape (Fig. 2i–k) or tothe cell monolayer (Fig. 2l) by tubular-like structures. Afterbeing cultured for more than 2 weeks, part of the gelledmixture appeared to crack or to become liquified and theculture had to be terminated.

Histological staining

HE staining of frozen sections of the 3D cultures revealedmany tubular-like structures with a centrally localized hollowlumen (Fig. 3a-e, a’-e’). Some of these tubular-like structureswere lined by a single layer of epithelial cells (Fig. 3a-d, a’-d’), which were similar to the secretory part of the eccrinesweat gland (Fig. 3f). Cells with a narrow top and wide bottomwere reminiscent of clear-like cells (Fig. 3, arrowhead) andcells with a wide top and narrow bottom resembled dark-likecells (Fig. 3, arrow); some of these tubular-like structures were

lined by stratified epithelium (Fig. 3e, e’), like the duct part(Fig. 3f). Immunohistochemical analysis demonstrated thatthe cultures expressed CK7 (Fig. 4a, a’), CK19 (Fig. 4b, b’),EMA (Fig. 4d) and actin (Fig. 4c).

Fig. 1 Eccrine sweat gland cells in two-dimensional (2D) culture. aIsolated human eccrine sweat gland tissue. b , c Cultured human eccrinesweat gland cells. c Tightly connected, cultured epithelial cells with theirtypical “cobblestone” morphology. Bars 100 μm

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Discussion

As is well known, humans secrete sweat to regulate bodytemperature (Saga 2002). In extensive third-degree burns,normal skin and its appendages are damaged and so

thermoregulatory function is impaired (Fu et al. 2006).Because the evaporation of sweat is the body’s primary mech-anism for heat loss, hyperthermia is generally thought to be aproblem for most burn patients. Although unburned areas ofthe patient’s body attempt to compensate by excessive

Fig. 2 Growth of eccrine sweatglands in matrigel and undermatrigel. Each cell (a) growing inmatrigel divided into 2 (b , c) ormore cells (d) and formed cellclusters (e–g , i–l), whereas cellsgrowing under matrigel onlyformed a standard monolayer (h).Note one cell cluster linked toanother cell cluster (i–k ; k is themerged image of i , j) or cellmonolayer (l) by tubular-likestructures. Bars 50 μm

Fig. 3 Hematoxylin and eosin (HE) staining of 3D cultures and humanskin tissues. Some tubular-like structures were seen with a single layer ofepithelial cells located peripherally and a lumen in the center (a–e , a’–e’).Some cells forming the tubular-like structures appeared in the shape ofpyramid. Cells with a narrow top and wide bottom were identified asclear-like cells (arrowhead) and those with a wide top and narrow bottomas dark-like cells (arrow); some cells forming the tubular-like structures

appeared in a stratified cuboidal epithelium composed of two layers ofcells (e , e’). HE staining of human skin tissues (f); the secretory parts ofthe eccrine sweat glands were lined by a simple pyramidal epithelium andthe ducts were lined by stratified cuboidal epithelium. Paired images aand a’ , b and b’ , c and c’ , d and d’ and e and e’ are the same, with thedashed lines in a’ , b’ , c’ , d’ and e’ showing the outline of the secretorypart or duct of the eccrine sweat glands. Bars 15 μm

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sweating, heat is produced faster than it can be dissipated,resulting in hyperthermia in patients, thereby reducing theskin’s adaptive capacity to the environment and affecting thequality of the burn patient’s life (Fu et al. 2006; Zhang and Fu2008; Li et al. 2006). Currently, no effective treatment existsfor patients with an irreversible loss of functional eccrinesweat glands caused by anhidrotic ectodermal dysplasia ormassive severe burns and so extensive research into the well-characterized skin with its functional skin appendages is im-portant (Zonana et al. 2000; Fu et al. 2006).

Traditional studies of cell and tissue regulation rely on theanalysis of cells grown in 2D cell-culture models. These 2Dmodels fail to reconstitute the in vivo cellular microenviron-ment and thus cannot maintain their differentiated functions.The 3D cell culture methods represent an attempt to solvethese limitations of 2D cultures.

Interactions between the extracellular matrix (ECM) andepithelial cells are necessary for the proper 3D organizationand function of the epithelium (Kozłowski et al. 2011).Matrigel basement membrane matrix (abbreviated tomatrigel), a solubilized basement membrane preparationextracted from Engelbreth-Holm-Swarm (EHS) mouse sarco-ma cells, is one of these recombinants (Kleinman and Martin2005). The components of matrigel include laminin, collagenIV, heparan sulfate proteoglycans, entactin and many growthfactors, such as transforming growth factor beta, EGF, insulin-like growth factor, fibroblast growth factor and tissue plas-minogen activator (Kleinman and Martin 2005). Matrigelpromotes the differentiation of many different cell types andthe outgrowth of differentiated cells from tissue explants.Cells growing in matrigel form 3D structures similar to thoseof the tissue of origin and so the differentiation response inmatrigel is dependent on the cell type (Kleinman and Martin2005). Inmatrigel, salivary-gland-derived cells form polarizedsecretory acinar-like structures, secrete acinar cell proteins,such as alpha-amylase, aquaporin-5, cytokeratins andmucin-1 and express tight junction markers, i.e., claudin-1,-2, -3 and -4, occludin, junctional adhesion molecule-A andzonula occludens-1 (Baek et al. 2012; Maria et al. 2011).Mammary epithelial cells grown in matrigel form 3D acinarstructures with a centrally localized hollow lumen structurallyresembling mammary alveoli in the functionally active mam-mary gland, express integrin receptors and tight junctionproteins and secrete beta-casein (Kozłowski et al. 2011).Vascular endothelial cells align and form a tape-like mono-layer and then change into a thinner tube-like structure with alumen (Okochi et al. 2009; Nathwani et al. 2010).

In the 3D culture of eccrine sweat glands in vitro, theepithelial cells formed tubular-like structures and coiled intosphere-like shapes reminiscent of the morphology of eccrinesweat glands in vivo. A sphere-like shape was sometimeslinked to another sphere-like shape or to the cell monolayerby tubular-like structures, which were different from eccrinesweat glands in vivo. In vivo, each eccrine sweat gland isindependent and opens directly onto skin surface (Saga 2002).Histological staining of the 3D cultures revealed tubular-likestructures with a centrally localized hollow lumen. Some ofthese tubular-like structures were lined by a single layer ofepithelial cells and some by stratified epithelium, which re-sembled the secretory part and duct part of the eccrine sweatgland in skin sections, respectively.

In previous studies, we have demonstrated that eccrinesweat glands express CK7, CK8, CK14, CK18, CK19,carcinoembryonic antigen (CEA), EMA, Ki67, p63, EGFand EGF receptor, with CEA only being expressed in sweatglands in normal skin (Li et al. 2009). In the 3D cultures,immmunohistochemical staining showed that the cells expressCK7, CK19, EMA and actin, markers of eccrine sweat glands(Li et al. 2009). Different from cells growing in matrigel, the

Fig. 4 Immunohistochemical analysis of 3D cultures. The 3D cultures inmatrigel express CK7 (a , a’), CK19 (b , b’), epithelial membrane antigen(EMA; d) and actin (c). Some tubular-like structures have a single layerof epithelial cells located peripherally and a lumen at the center (a , a’ , b ,b’). Nuclei are stained blue (DAPI). The immunostaining of CK7, CK19,EMA and actin is in red . Negative control (e). Paired images a and a’and b and b’ are the same, with the dashed lines in a’ and b’ showing theoutline of the secretory part of the eccrine sweat glands. Bars 10 μm

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cells growing under the matrigel do not form a 3D structurebut rather the standard cell monolayer. Under 3D cultureconditions, eccrine sweat gland cells are more differentiatedthan when they are cultured on plastic substrates.

In conclusion, eccrine sweat gland cells cultured in matrigelform 3D structures that mimic the morphology of eccrine sweatglands in vivo. This kind of 3D culture mode enhances theexpression of differentiated functions, improves tissue organiza-tion and is valuable in allowing further insights into the devel-opment, wound repair and regeneration of eccrine sweat glandsand into the diseases associated with eccrine sweat glands.However, we still have a long way to go to reconstitute thefeatures of a living eccrine sweat gland completely. After all,the living eccrine sweat gland is composed of many cells that arehighly dynamic in terms of their polarity, mechanical propertiesand biochemical microenvironment.

References

Baek H, Noh YH, Lee JH, Yeon SI, Jeong J, Kwon H (2012)Autonomous isolation, long-term culture and differentiation poten-tial of adult salivary gland-derived stem/progenitor cells. J TissueEng Regen Med. doi:10.1002/term.1572

Cai S, PanY, HanB, Sun TZ, Sheng ZY, FuXB (2011) Transplantation ofhuman bone marrow-derived mesenchymal stem cells transfectedwith ectodysplasin for regeneration of sweat glands. Chin Med J(Engl) 124:2260–2268

Fu X, Li X, Cheng B, Chen W, Sheng Z (2005) Engineered growthfactors and cutaneous wound healing: success and possible ques-tions in the past 10 years. Wound Repair Regen 13:122–130

Fu X, Qu Z, Sheng Z (2006) Potentiality of mesenchymal stem cells inregeneration of sweat glands. J Surg Res 136:204–208

Grice EA, Segre JA (2011) The skin microbiome. Nat Rev Microbiol9:244–253

Kleinman HK, Martin GR (2005) Matrigel: basement membrane matrixwith biological activity. Semin Cancer Biol 15:378–386

Kozłowski M, Wilczak J, Motyl T, Gajewska M (2011) Role of extracel-lular matrix and prolactin in functional differentiation of bovineBME-UV1 mammary epithelial cells. Pol J Vet Sci 14:433–442

Li H, Fu X, Ouyang Y, Cai C,Wang J, Sun T (2006) Adult bone-marrow-derived mesenchymal stem cells contribute to wound healing of skinappendages. Cell Tissue Res 326:725–736

Li HH, Zhou G, Fu XB, Zhang L, Sun TZ (2009) Antigen expression ofhuman eccrine sweat glands. J Cutan Pathol 36:318–324

Maria OM, Maria O, Liu Y, Komarova SV, Tran SD (2011) Matrigelimproves functional properties of human submandibular salivarygland cell line. Int J Biochem Cell Biol 43:622–631

Nathwani SM, Butler S, Meegan MJ, Campiani G, Lawler M, WilliamsDC, Zisterer DM (2010) Dual targeting of tumour cells and hostendothelial cells by novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines. Cancer Chemother Pharmacol 65:289–300

Okochi N, Okazaki T, Hattori H (2009) Encouraging effect of cadherin-mediated cell-cell junctions on transfer printing of micropatternedvascular endothelial cells. Langmuir 25:6947–6953

SagaK (2002) Structure and function of human sweat glands studiedwithhistochemistry and cytochemistry. Prog Histochem Cytochem37:323–386

Sheng Z, Fu X, Cai S, Lei Y, Sun T, Bai X, Chen M (2009) Regenerationof functional sweat gland-like structures by transplanted differenti-ated bone marrow mesenchymal stem cells. Wound Repair Regen17:427–435

Shibasaki M, Wilson TE, Crandall CG (2006) Neural control and mech-anisms of eccrine sweating during heat stress and exercise. J ApplPhysiol 100:1692–1701

Zhang CP, FuXB (2008) Therapeutic potential of stem cells in skin repairand regeneration. Chin J Traumatol 11:209–221

Zonana J, Elder ME, Schneider LC, Orlow SJ, Moss C, Golabi M,Shapira SK, Farndon PA, Wara DW, Emmal SA, Ferguson BM(2000) A novel X-linked disorder of immune deficiency andhypohidrotic ectodermal dysplasia is allelic to incontinentiapigmenti and due to mutations in IKK-gamma (NEMO). Am JHum Genet 67:1555–1562

902 Cell Tissue Res (2013) 354:897–902