Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium.
OBJECTIVE
To investigate the effect of FGF-2 on corneal endothelial cell survival in porcine and human corneas during corneal storage in a serum-free medium.


METHODS
Porcine and paired human corneas were stored at 32 degrees C for 9 and 22 days, respectively. One cornea of each pair was stored in a serum-free culture medium, and the mate was preserved in the same medium supplemented with 10 ng/mL FGF-2. Quantitative analysis of corneal damage after storage was determined by the Janus green photometry technique. 5-Bromo-2-deoxyuridine (BrdU) labeling of the endothelium determined the effect of FGF-2 on endothelial proliferation during storage. Additional cell culture studies were performed to elucidate the role of FGF-2 on the incidence of endothelial apoptosis after serum deprivation.


RESULTS
When FGF-2 was added to the serum-free medium, the damage rates of porcine endothelia were reduced from 15.1% +/- 8.7% (control) to 6.4% +/- 2.0% after 9 days and from 25.3% +/- 10.2% to 13.6% +/- 4.2% after 22 days of storage. In the human corneas stored during 22 days in FGF-2-supplemented medium, the amount of endothelial damage was 11.8% +/- 3.2%, which was significantly less damage than in the control fellow corneas stored in unsupplemented serum-free medium (19.3% +/- 6.3%; P < 0.01). DNA synthesis was not enhanced in corneas stored in serum-free medium, serum-free medium+FGF-2, or medium containing 10% FCS. Only a few (3.8%) TUNEL-positive endothelial cells were detected in cultures maintained in FGF-2-supplemented serum-free medium compared with a high number (48%) of apoptotic cells in control cultures.


CONCLUSIONS
FGF-2 efficiently reduces human corneal endothelial damage that occurs during organ culture storage in a serum-free medium. This effect is truly protective, because no proliferative activity and a decreased rate of apoptosis were determined. FGF-2 emerges as an important component of a future serum-free corneal organ-culture medium established to replace fetal calf serum (FCS) as a potential source of recipient infection.
Rieck
Peter W
PW
Department of Ophthalmology, Charité Medical Faculty, Humboldt University Berlin, Germany. peter.rieck@charite.de
Gigon
Marcel
M
Jaroszewski
Jan
J
Pleyer
Uwe
U
Hartmann
Christian
C
D000368:Aged; D000818:Animals; D017209:Apoptosis; D001391:Azo Compounds; D001973:Bromodeoxyuridine / Q000378:metabolism; D002455:Cell Division; D002470:Cell Survival / Q000502:physiology; D003315:Cornea; D016895:Culture Media, Serum-Free; D019610:Cytoprotection / Q000187:drug effects; D004261:DNA Replication; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D015590:Fluorophotometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D008875:Middle Aged; D009924:Organ Culture Techniques; D009926:Organ Preservation; D011994:Recombinant Proteins; D013552:Swine; D013997:Time Factors
D001391:Azo Compounds; D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; C047446:janus green; D001973:Bromodeoxyuridine