Establishment of a human lacrimal gland epithelial culture system with in vivo mimicry and its substrate modulation.
OBJECTIVE
Viable single human lacrimal gland (LG) epithelial cells were obtained by serial incubation in chelating and enzymatic solutions. These cells were evaluated for outgrowth in growth factor-enriched medium using the following culture substrates: Matrigel type I collagen gel with or without fibroblasts, and plastic. Each epithelial outgrowth was characterized morphologically and immunohistochemically, and their growth and viability were examined by bromodeoxyuridine (BrdU) labeling and a quantitative cell viability assay. Synthesized proteins were evaluated by enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gradient gels, and 14C-amino acid incorporation.


RESULTS
LG epithelial cells plated on Matrigel formed clusters with central cavities that contained lactoferrin, mimicking acinar complexes in vivo. Cells plated on Matrigel containing fibroblasts formed tubuloacinar structures and resembled the human LG in vivo. Cells plated on collagen gel or collagen gel containing fibroblasts formed islands or a monolayer, and lactoferrin was detected in incomplete cavities of epithelia on the latter substrate. Epithelial cells plated on plastic formed a monolayer, and cellular expression of lactoferrin was weak and sporadic. Cellular release of lactoferrin measured by ELISA supported the results of immunohistochemistry. Significant differences in proliferative rate were noted between substrates; cells grown on plastic had the highest proliferative rate, whereas cells grown on Matrigel had the lowest rate.


CONCLUSIONS
Culture substrates modulate LG epithelial cell morphology, proliferative rate, and production of the tear protein lactoferrin. Matrigel promotes acinar differentiation to a greater extent than collagen gel and plastic. Incorporation of fibroblasts in substrates promotes ductal differentiation.
Yoshino
K
K
Yoshino Eye Clinic and Department of Ophthalmology, Tokyo Dental College, Chiba, Japan.
D000293:Adolescent; D000328:Adult; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D003094:Collagen; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005136:Eye Proteins / Q000096:biosynthesis; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007765:Lacrimal Apparatus / Q000166:cytology* / Q000378:metabolism; D007781:Lactoferrin / Q000096:biosynthesis; D008297:Male
D005136:Eye Proteins; C042434:tear proteins; D003094:Collagen; D007781:Lactoferrin