[Preliminary study on formation of eccrine sweat gland-like structure in three-dimensional cell culture].
OBJECTIVE
To explore a method to reconstruct eccrine sweat gland-like structure in vitro.


METHODS
Isolated from the normal axillary full-thickness skin donated by volunteers sweat gland epithelial cells were cultured in vitro and were observed under inverted phase contrast microscope. These cells at the density of 2 x 10(5)/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-like structure was observed by confocal laser scanning microscope, HE staining and immunohistochemistry staining.


RESULTS
Primary epithelial cells in the secretory portion of sweat gland were attached and spindle-shaped 24 hours after inoculation, and were under polyclonal grain-like growth 2-3 days thereafter. Cobblestone-like 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 proliferation was evident. For group C, cell division and proliferation occurred 2-3 days after three-dimensional culture; the proliferated 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.


CONCLUSIONS
The sweat gland epithelial cells can be induced to form eccrine sweat gland-like structure through three-dimensional culture in Matrigel.
Zhou
Lifeng
L
Department of Dermatology, Daping Hospital, Third Military Medical University, Wound Healing and Tissue Engineering Institute of Chongqing Municipality, Chongqing, 400042, P.R. China.
Wu
Jinjin
J
Lei
Xia
X
Lu
Rong
R
Lu
Yuangang
Y
Tang
Shuqian
S
D018929:Cell Culture Techniques; D002455:Cell Division; D002478:Cells, Cultured; D003094:Collagen; D004338:Drug Combinations; D004847:Epithelial Cells / Q000166:cytology*; D006801:Humans; D007797:Laminin; D011509:Proteoglycans; D013545:Sweat Glands / Q000166:cytology*
D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen