The use of autologous serum in the development of corneal and oral epithelial equivalents in patients with Stevens-Johnson syndrome.
OBJECTIVE
To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS).


METHODS
AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry.


RESULTS
BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy.


CONCLUSIONS
AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.
Nakamura
Takahiro
T
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Graduate School of Medicine, Hirokoji, Kyoto, Japan. tnakamur@ophth.kpu-m.ac.jp
Ang
Leonard P K
LP
Rigby
Helen
H
Sekiyama
Eiichi
E
Inatomi
Tsutomu
T
Sotozono
Chie
C
Fullwood
Nigel J
NJ
Kinoshita
Shigeru
S
D000328:Adult; D000368:Aged; D001485:Basement Membrane / Q000648:ultrastructure; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D049109:Cell Proliferation; D017690:Cell Transplantation; D003114:Colony-Forming Units Assay; D003229:Conjunctival Diseases / Q000097:blood*; D003315:Cornea / Q000166:cytology* / Q000378:metabolism; D003470:Culture Media; D004247:DNA / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007633:Keratins / Q000378:metabolism; D008297:Male; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D009061:Mouth Mucosa / Q000166:cytology* / Q000378:metabolism; D044967:Serum / Q000502:physiology*; D013262:Stevens-Johnson Syndrome / Q000097:blood*
D003470:Culture Media; D007633:Keratins; D004247:DNA