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Keywords:

  • tubular;
  • morphogenesis;
  • eccrine sweat gland;
  • epithelial cell;
  • matrigel

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Human eccrine sweat glands are tubule-structured glands of the skin that are vital in thermoregulation, secretion, and excretion of water and electrolytes. A study of tubular morphogenesis in vitro would facilitate the development of a tissue engineering model for eccrine sweat glands and other tubule-structured glands. Matrigel, a basement membrane matrix, has been shown to promote differentiation and morphogenesis of many different cell types, including tubular cells. This study investigated the growth, differentiation, and tubular morphogenesis of human eccrine sweat gland epithelial cells cultured in Matrigel. Human eccrine gland epithelial cells were isolated and cultured in vitro. The cell growth in Matrigel was evidenced by the formation of cell clusters, which were observed under an inverted microscope. The internal structure of the cell clusters was further investigated by hematoxylin–eosin (HE) staining and confocal laser scanning microscopy (CLSM) of propidium iodide-stained nuclei. The results demonstrated that although on a plastic surface or in a collagen gel the cells could not form tubular structures, they formed tubular structures when cultured in Matrigel. Consequently, we conclude that Matrigel can promote tubular morphogenesis of human eccrine sweat gland epithelial cells. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

The importance of the human sweat gland has long been recognized because of its vital role in thermoregulation, secretion, and excretion (Servetnyk et al., 2007). In vitro cultivation of normal human sweat gland epithelial cells and studies on the three-dimensional reconstruction of eccrine sweat glands have potential biological and clinical significance. A thorough understanding of how these cells and glands develop is essential to gain further insights into the epithelial development mechanisms and diseases associated with sweat glands or other tubular organs.

Studies of the human eccrine sweat gland have been hindered by the difficulties involved in gland isolation and cell cultivation. Earlier studies used microdissection and tissue culture techniques, both of which were extremely difficult with low yields (Lee et al., 1989; Reddy et al., 1992; Mork et al., 1995). As a result, a simple method of shearing and dissection of skin biopsies by repetitive cutting to release eccrine sweat glands was developed (Lee et al., 1989). Several laboratories have reported the use of different culture methods for sweat gland epithelial cells, including tissue explants and collagenase digestion (Brayden et al., 1995; Li et al., 2005; Lei et al., 2008). For example, a neutral red staining was used in Brayden's method to highlight the sweat gland, thus making harvesting of cells more efficient than previous methods. (Brayden et al., 1995). We have also reported an effective culture method for sweat gland cells (Lei et al., 2008) and have used this method in this study to investigate their tubular morphogenesis.

Epithelial tubular morphogenesis is the basis for organizing the functional units of parenchymal organs (Barka et al., 2005). Tubular morphogenesis is also related to cancer invasion and metastasis. Tubular branching observed in cell culture in vitro is a complex morphogenic process that depends on tight coordination of cell replication, polarity and movement, rearrangements of the cytoskeleton and intercellular junction complexes, and interaction with and remodeling of the extracellular matrix (ECM; Hogan et al., 2002; Zegers et al., 2003).

Developmental interactions between the ECM and the epithelium induce both the morphogenesis and the cytodifferentiation of epithelial cells (Schwartz et al., 1999). It was observed that during embryonic development, along with the degradation and rebuilding of ECM, the eccrine sweat gland bud develops into gland coil and duct (Loomis 2001). Thus, ECM not only provides support and scaffolding for cells but also serves as a 3D substructure for cell adhesion and movement, a storage depot for growth factors, chemokines and cytokines, and a signal for morphogenesis and differentiation. Matrigel is an extract derived from Engelbreth-Holm-Swarm tumor that contains all the basement membrane proteins and has been shown to promote differentiation and morphogenesis of many different cell types (Kleinman et al., 2005). Cells on or in this matrix usually associate with each other in three dimensions, and then form structures similar to those of the original tissues. For example, both established and primary mammary epithelial cells would form ducts, ductules, and gland-like structures, which secrete casein into the lumen (Li et al., 1987). Furthermore, chondrocytes were found to form cartilaginous nodules on the surface of the matrix (Bradham et al., 1995); endothelial cells would attach and align in 1 hr and form capillary-like structures with a lumen overnight (Kubota et al., 1988); salivary gland cells would form acinar-like structures and produce amylase when cultured in Matrigel (Hoffman et al., 1996).

In this study, human eccrine sweat gland cells were harvested by collagenase digestion and cultured in keratinocyte serum-free medium (KSFM). The cells developed into a cluster after culture in Matrigel, which became increasingly bigger during the first 9 days. The tubular structures which are characterized by a single layer of epithelial cells and a central lumen were stained with hematoxylin–eosin (HE) and observed under a confocal laser scanning microscopy (CLSM). A control group consisted of cells cultured in collagen that were not able to form cell clusters.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Cell Culture

The skin specimens were obtained from healthy individuals undergoing axilla plastic surgery. Ethical permission was granted by the Ethics Committee of Daping Hospital (Chongqing, China), and informed consent was obtained from patients. The cell culture method used has been described previously (Lei et al., 2008). Briefly, the gland coils were stained by neutral red and digested by type II collagenase (0.2%, Gibco BRL) at 37°C for ∼6 hr, then separated by centrifugation. The harvested cells were resuspended in defined KSFM (Gibco BRL), counted, and seeded (∼1–5 × 104 viable cells/cm2) on collagen-coated plastic dishes. Cell viability was measured by trypan blue staining. The cells were fed every other day and incubated in a humidified environment with 5% carbon dioxide at 37°C. The cells were observed daily under an inverted microscope (Olympus, Japan). Harvested cells were identified by immunocytochemistry as previously described (Lei et al., 2008).

As some fibroblasts and hair papilla cells could grow during culture, elimination of contaminant cells was achieved by repeated trypsinization. After 1 mL of 0.25% trypsin (Amresco, OH) was added to 25 cm2 dishes, the cells were observed under an inverted microscope. Fibroblast and hair papilla had rounded up, while epithelial cells showed no change. The Dulbecco's modified eagle's medium containing serum was then added to inactivate trypsin. After swilling with D-Hank's twice, the cells were cultured in KSFM sequentially. Trypsinization could be repeated during the culture of cells.

Three-Dimensional Culture in Matrigel or Collagen

Aliquots (0.2 mL) of cold Matrigel solution (BD Biosciences, Bedford, MA) or Collagen I (Invitrogen, Carlsbad, CA) diluted with cold KSFM at a 1:1 ratio were added to the chambers of a 48-chamber Falcon Culture Slide (Fisher Scientific, Pittsburgh, PA), followed by the addition of human eccrine sweat gland epithelial cells (5 × 103 cells) in diluted Matrigel or Collagen I (0.2 mL). To promote gel formation, the slides were incubated at 37°C.

Cell Cluster Growth

The cell clusters grown in Matrigel or Collagen I were observed under an inverted microscope, and the diameters of cell clusters were measured daily using an ocular micrometer (Olympus, Tokyo, Japan), with the cultures photographed on Days 1, 3, 5, 7, 9, 11, and 13. The diametric data were compared by ANOVA (Analysis of variance) test and t test and analyzed by SPSS 11.0 software. Results were expressed as the mean ± SEM, and P < 0.05 was considered to be statistically significant.

Histological Techniques

After sweat gland epithelial cells were cultured in Matrigel for 9 days, 30-μm frozen sections of Matrigel were prepared for histological observation. Frozen sections were affixed to a polyfrost glass slide (Fisher, MA). After HE staining, the internal structures of the cell cluster were observed under an inverted microscope.

Confocal Microscopy

First, the cell clusters were stained with propidium iodide (1:50 dilution; Boehringer Mannheim, MD) and the resulting integrated shape was photographed by confocal microscope (Leica, German). Then frozen sections (30 μm) were also prepared for confocal tomoscan analysis. For the frozen sections, cell nuclei were stained with propidium iodide for 1 min and then observed under confocal microscope at low and high magnifications (×100 and ×500). The excitation wavelength was 428 nm, and the emission wavelength was 600 nm. The stepping tomoscan was performed from bottom to surface to observe the inner structure of the cell cluster. Finally, a 3D reconstruction of the confocal images was performed using Leica confocal software.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Cell Culture

After staining with neutral red, the sweat glands were pale red in color under a dissecting microscope (×20) and were twisted together like an irregular ball (Fig. 1A).

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Figure 1. Cell culture of human eccrine sweat gland(A–D). (A) Morphology of eccrine gland (20×). (B) Before trypsinization. Trypan blue exclusion test, Arrow indicates a nonviable cell has taken up trypan blue (200×, scale bar = 200 μm). (C) Before trypsinization. Contamination by some long fusiform fibroblasts and hair papilla cells (arrows) (200×, scale bar = 100 μm). (D) After trypsinization. Primary culture of sweat gland epithelial cells, which have reached confluency. (200×, scale bar = 100 μm).

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Harvested cells were seeded on culture dishes. More than 90% of the harvested cells were alive according to the trypan blue exclusion test(Fig.1B). Twenty-four hours after seeding, the cultured cells attached to the base of the culture dish. They were predominantly small and shaped like wheat kernels. The cells started to grow and extended to form small epithelial islets from Day 4 to Day 5. Initially, both fibroblasts and hair papilla cells exhibited a long fusiform shape (Fig. 1C). After being digested with trypsin, these cells rounded up and were washed out by D-Hank's. After several rounds of trypsinization, no fibroblast or hair papilla cells were left to contaminate the sweat gland epithelial cells (Fig. 1D).

Primary cultures were confluent after 3 weeks and exhibited a “cobblestone” appearance under the phase contrast microscope (Fig. 1D). Confluent cells appeared to be flat and polygonal with a medial round nucleus.

Growth of Cell Clusters in Matrigel

Cells grew and differentiated quickly in Matrigel. The diameters of the cell clusters were measured by an ocular micrometer every other day, and the results were analyzed statistically (Table 1). Initially, single cells appeared to form cell clusters with a cyst-like structure; then the diameter and the number of cells per cluster increased. We found that the cells had an overall doubling time of 48 hr in Matrigel during the first 11 days. During the culturing process, the cell clusters increased from ∼2–3 cells on Day 3 to more than 20 on Day 9 (Fig. 2A–D). After culturing for 11 days, many cell clusters had appeared to form an irregular ball, which resembled a miniature version of the eccrine sweat gland in vivo. (Fig. 2E,F). In contrast, the cells in the control group were still round on Day 3 (Fig. 2G) and appeared as if they were just seeded in collagen, and could not grow into cell clusters even after 11 days (Fig. 2H).

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Figure 2. Eccrine sweat gland epithelial cells cultured in matrigel (A–F) or on collagen (G and H). (A, B, and G) 3 days culture. (C and D) 9 days culture. (E, F, and H) 11 days culture. Arrows indicate cell clusters. (A, C, E, G, and H 100×, scale bar = 100 μm, B, D, and F 400×, scale bar = 25 μm).

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Table 1. Diameters of cell clusters in Matrigel from 3d to 13d after the cells were cultured in Matrigel
Culture time (day)35791113
Diameter of cell cluster (μm)14.7 ± 5.0624.4 ± 5.1250.83 ± 9.9282.78 ± 20.02115.28 ± 20.13132.50 ± 29.42

Histological Observation

When stained with HE, the cytoplasm of cell was pink and the nuclei blue. Tubular structures were found with a single layer of epithelial cells located peripherally and a lumen formation centrally (Fig. 3A–D). Some polarized cells with narrow top and wide bottom might be the dark cells (Fig. 3 arrow).

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Figure 3. HE staining of cell clusters in Matrigel. (A–D) Four cell clusters seen after 9 days culture in Matrigel. Tubular structures with a single layer of epithelial cells located peripherally and a central lumen are present. The arrow indicates a cell with a narrow top and wide bottom, which could be a dark cell. (scale bar = 30 μm).

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Confocal Microscopy

After staining with propidium iodide, the cell nuclei were marked by intense red florescence. Cell clusters with a cyst-like structure were observed by confocal microscopy, which showed that some clusters have tubular structures while cavities in the cysts were also discovered. To further study the 3D structure, we also prepared 30-μm frozen sections of Matrigel with human eccrine sweat gland epithelial cell clusters for confocal microscopy. The internal structures of cysts were scanned by confocal microscopy every 2–5 μm for each layer. Three-dimensional reconstructions were performed and analyzed using confocal software.

After 9 days of culture in Matrigel, branching tubular structures were present in the cultures. The lumen of a single branch cord was surrounded by a monolayer of cells (Fig. 4A,C,E,G). Photos of the tubular structures taken under phase contrast microscopy (Fig. 4B,D,F,H) showed the longitudinal and cross sections of tubular structures (Fig. 4A,C,E), and cyst-like structures containing cavities (Fig 4G) with an elongated or branched structure.

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Figure 4. Observation of cell clusters with confocal laser (A–H). Four cell clusters observed with confocal laser (A, C, E, and G) and the same clusters observed with phase contrast microscopy (B, D, F, and H). The lumen of a single tubule is surrounded by a monolayer of cells.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

We have demonstrated a culture method that involves enriching human eccrine sweat gland epithelial cells by enzymatic digestion followed by culture in Matrigel, which promotes their differentiation into cyst-like cell clusters with an internal tubular structure. Tubular morphogenesis is a fundamental developing process during organogenesis that requires mutual interactions of epithelial and mesenchymal elements. A recent study on the formation of branched tubules by different epithelial cells and by endothelial cells in vitro has been reported (Zegers et al., 2003), but the mechanisms of acinus formation and tubular morphogenesis have not been fully probed and analyzed. Matrigel has been shown to promote differentiation and morphogenesis of many different cell types (Nicosia et al., 1990). Various models of differentiation have been developed, some of which have led to widely used assays, such as the capillary-like tube formation for angiogenesis (Nicosia et al., 1990; Auerbach et al., 2003). Once differentiated, cells can be transplanted back into animals for organ replacement/repair. Hence, differentiated cells are potentially useful for tissue engineering and wound repair applications. Although the morphological behavior of cells within a Matrigel basement membrane is complex, Matrigel can provide an extracellular growth environment in vitro, similar to the more important and relevant in vivo environment in contrast to collagen, which could not provide necessary ECM for cell differentiation. Other factors such as integrins, proteins (laminin, collagen, and proteoglycan), and growth factors [Epidermal growth factor (EGF), Hepatocyte growth factor (HGF), Transforming growth factor (TGF), and Platelet-derived growth factor (PDGF)] all play important roles in cell differentiation (Barka et al., 2005; Sodunke et al., 2007), but the complete signaling mechanism on which the morphogenesis depends remains unknown.

There are two elements for tubular morphogenesis in vitro: the cells that form the layer and the lumen formation. In this study, eccrine sweat gland epithelial cells in Matrigel grew into a cell cluster with a cyst-like structure, followed by development of tubular structures. The cells had an overall doubling time of 48 h in Matrigel during the first 11 days. In addition, tubular structures characterized by a single layer of epithelial cells and a central lumen were identified by HE staining and CLSM. The tubular structures consisted of a single layer of cells and a central cavity. The cells in Collagen I (control group) were grown in 3D conditions but did not form clusters and tubules.

The tubular morphogenesis of human eccrine sweat epithelial cells in Matrigel further demonstrates the effect of Matrigel on cell differentiation. Our future studies will focus on the mechanism of cell differentiation in ECM, the possibility of reconstructing the eccrine sweat gland coils and ducts in tissue-engineering skin, and the recovery of sweat excretion.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

This study was supported by National Natural Foundation of China (30801193), and National 863 Project of China (2006AA02A121). The authors acknowledge the research grant from Professor Jinjin Wu. The authors are also grateful to Professor Zaiyun Long from the Field Surgery Institute of the Third Military Medical University for the confocal microscopy work.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED