Multi-layered culture of primary human conjunctival epithelial cells producing MUC5AC.
The purpose of our study was to establish a system for culturing normal human conjunctival epithelial (NHCE) cells under serum-free culture conditions without compromising their ability to differentiate into a mucous epithelium. To this end, small pieces of normal conjunctiva were biopsied from patients undergoing cataract surgery. Obtained NHCE cells were cultured in bronchial epithelial growth medium (BEGM) under serum free culture conditions and passage 3 cells were air-lifted. Cultured NHCE cells displayed typical epithelial morphology. Expression of cytokeratin 19 and conjunctival epithelial specific carbohydrate residue were detected. Air-lifted NHCE cells demonstrated an increase in stratification and differentiation into goblet cells up to 3weeks under air-liquid interface (ALI) culture condition. NHCE cells expressed MUC1, MUC4, MUC16, and MUC5AC mRNA, and MUC5AC production and secretion increased in a time dependent manner after culture under ALI conditions. Exposure of cells to proinflammatory cytokines (TNF-alpha or IFN-gamma) resulted in upregulation of MUC1, MUC4, MUC16, and MUC5AC gene expression. In conclusion, differentiated NHCE cells showed features of a multi-layered conjunctival epithelium, including goblet cells, and retained functional characteristics similar to those seen in vivo. This cell culture system can better facilitate investigation of conjunctival epithelial cell biology and goblet cell differentiation.
Chung
So-Hyang
SH
Department of Ophthalmology, Inje University College of Medicine, SeoulPaik Hospital, Seoul, Republic of Korea.
Lee
Joon H
JH
Yoon
Joo-Heon
JH
Lee
Hyung Keun
HK
Seo
Kyoung Yul
KY
D000328:Adult; D000368:Aged; D018394:CA-125 Antigen / Q000096:biosynthesis / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D049109:Cell Proliferation; D048429:Cell Size; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D016207:Cytokines / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure; D005136:Eye Proteins / Q000096:biosynthesis*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D020397:Goblet Cells / Q000166:cytology; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D008875:Middle Aged; D055271:Mucin 5AC; D018396:Mucin-1 / Q000096:biosynthesis / Q000235:genetics; D009077:Mucins / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
D018394:CA-125 Antigen; D016207:Cytokines; D005136:Eye Proteins; D018836:Inflammation Mediators; C060192:MUC1 protein, human; C451759:MUC16 protein, human; C524678:MUC5AC protein, human; D008565:Membrane Proteins; D055271:Mucin 5AC; D018396:Mucin-1; D009077:Mucins; D012333:RNA, Messenger