Object ive To explore a method to recons t ruct eccr ine sweat gland- l ike s t ructure in vitro. Methods Isolated from the normal axillary full-thickness skin donated by volunteers sweat gland epithel ial cells were cultured in vitro and were observed under inverted phase contrast microscope. These cells at the density of 2 × 105/cm2 were inoculated underneath the Matrigel (group A), on the top of the Matrigel (group B) and in the Matrigel (group C), respectively,for three-dimensional culture. The formation of eccrine sweat gland-l ike structure was observed by confocal laser scanning microscope, HE staining and immunohistochemistry staining. Results Primary epithel ial cells in the secretory portion of sweat gland were attached and spindle-shaped 24 hours after inoculation, and were under polyclonal grain-l ike growth 2-3 days thereafter. Cobblestone-l ike appearances of these cells were evident 14 days after inoculation and the confluent cells were flat and polygonal with relatively big round cell nucleus. Morphologically, subcultured cells at passage 1 were similar to the primary cells; cells at passage 2 were irregular and most of them had long pseudopodium; cells at passage 3 were star-shaped and big and had fusion with adjacent cells. For group A, tubular structure was formed 11 days after three-dimensional culture. For group B, stretched and filamentous-shaped cytoplasm was observed 8 hours after three-dimensional culture, with the formation of lumen or half-lumen structure, but no significant prol iferation was evident. For group C, cell division and prol iferation occurred 2-3 days after three-dimensional culture; the prol iferated cells were closely arranged into tubular structure with obvious lacunae in the middle, which gradually developed into irregular ball-shaped structure with the increase of neonatal cells. The laser scanning confocal microscope observation showed the formation of spherical structure in group C, with tubular structure in the center of cell mass; HE staining testified the spherical structure in group C was tubular structure. The immunohistochemistry staining demonstrated keratin 18 and carcinoembryonic antigen were positively expressed in group C, which was similar to the tubular structure of secretory portion of sweat gland. Conclusion The sweat gland epithel ial cells can be induced to form eccrine sweat gland-l ike structure through three-dimensional culture in Matrigel.
To investigate the effect of hepatocyte growth factor (HGF) on prol iferation of cultured human eccrine sweat gland epithel ial cells (hESGc) and the involvement of phosphorylation of ERK1/2. Methods hESGc were cultured in keratinocyte serum free medium (KSFM) and the first generation of hESGc was harvested. The expression of C-met was detected by immunocytochemistry. MTT assay was used to detect the effect of HGF on the prol iferation of hESGc. The cells were divided into blank group, control group and experimental group. The culture density was 2 × 103 cells/hole in control group and experimental group. Two hundred μL KSFM with HGF in different levels was added to every hole. hESGcwere cultured in KSFM with HGF at different levels (2, 20, 40 and 80 ng/mL) in experimental group, in KSFM without HGF incontrol group, and in KSFM without HGF and no hESGc in blank group. The cell prol iferation was observed in xperimental group 2 and 4 days later. Western blot was used to detect the expression of phosphorylated ERK1/2 at 40 ng/mL HGF after 0, 5, 30, 90 and 120 minutes. Results The results were positive for anti-C-met staining in the cytoplasm. HGF (40 ng/mL and 80 ng/mL) significantly improved the prol iferation of hESGc (P lt; 0.05). When cultured in the KSFM with 40 ng/mL HGF, the cell prol iferation rate and the absorbance were 74.2%, 0.239 3 ± 0.070 9 at 2 days and 74.8%, 0.287 8 ± 0.074 3 at 4 days; showing significant differences when compared with control group (P lt; 0.05). When cultured in KSFM with 80 ng/mL HGF, the cell prol iferation rate and the absorbance were 54.5%, 0.212 3 ± 0.059 2 at 2 days and 40.3%, 0.231 0 ± 0.056 7 at 4 days; showing significant differences when compared with control group (P lt; 0.05). The expression of p-ERK1/2 reached to the maximum after stimulation of 40 ng/mL HGF for 5 minutes, and relative integral absorbance (RIA) was 0.593 2 ± 0.192 2, increased 8.1 times compared with instant stimulation (P lt; 0.01). Conclusion HGF could induce the prol iferation of hESGc and activate the phosphorylation of ERK1/2 protein.
ObjectiveTo investigate the three-dimensional (3D) culture and morphology of human eccrine sweat gland cells. MethodsThe human eccrine sweat gland cells were isolated from normal abdominal full thickness skin by digestion of type II collagenase, and cultured in defined-keratinocyte serum free medium supplemented with 5 ng/mL recombinant human epidermal growth factor, 25 mg/mL bovine pituitary extract, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37℃ in a humidified atmosphere of 5%CO2/95% air incubator. When the cell fusion reached above 80%, the cells were harvested and the concentration was adjusted to 1×105 cells/mL. The mixture of 0.3 mL cell suspension and 0.3 mL Matrigel basement-membrane matrix was cultured in 12-well plate. The cell growth was observed under an inverted phase contrast microscope. At 14 days after culture, frozen sections were prepared and were stained with HE to observe the cells morphology, and immunohistochemical analysis was used to detect the antigen expressions of cytokeratin 7 (CK7) and CK19. ResultsInverted phase contrast microscope observation showed that many free eccrine sweat gland tissues were seen after digestion of type II collagenase; eccrine sweat gland cells grew adhering to the wall at 3-5 days and continued division for 2-3 weeks to form single ring around the block sweat glands; cellular senescence were observed after 3-4 weeks. During the process of 3D culture, the single eccrine sweat gland cell divided into 2-4 cells after 2-3 days, and these cells subsequently formed small cell clusters, tubular-like structures and finally spheric-like shapes. After cultured for about 2 weeks, there was crack in part of the gelled mixture or liquefaction occurred. HE staining of frozen sections of the 3D cultures showed some of the tubular-like structures composed of 1-2 layers of epithelial cells, which were similar to the secretion part and the duct part of the eccrine sweat gland. Immunohistochemical analysis showed that CK7 and CK19 antigens expressed positively in the cells. ConclusionHuman eccrine sweat gland cells cultured in Matrigel can form the 3D structures which simulate the morphology of eccrine sweat glands in vivo.