Purchase this article with an account.
Lucas Vianna, Hao Dong Li, Jeffrey D Holiman, Chris Stoeger, Rubens Belfort, Albert S Jun; . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1160.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The use of human serum supplemented media (HS-SM) for human corneal endothelial cell (HCEC) culture has been recently reported by our group, with potential advantages over fetal bovine serum supplemented media (FBS-SM) towards making cell therapy feasible for endothelial diseases in clinical practice. The maintenance of endothelial cell characteristics after cryopreservation would be another important consideration regarding this approach. This study compares cryopreserved HCECs grown in HS-SM to cryopreserved HCECs grown in FBS-SM.
Three pairs of human corneas from donors aged 8, 28, and 31 years old were obtained from Lions VisionGift (Portland, OR). From each pair, one cornea was used to start a HCEC culture with HS-SM and the other cornea with FBS-SM. Upon reaching confluence (P0), the 6 cell populations were frozen using 10% dimethyl sulfoxide containing medium. Thawed cells grown in HS-SM were compared to thawed cells grown in FBS-SM by morphology, growth curve, and protein (immunohistochemistry) and gene expression (real time-reverse transcriptase polymerase chain reaction - RT-PCR) for zonula ocludens-1 (ZO-1, gene TJP1), sodium/potassium ATPase (Na+/K+-ATPase, gene ATP1A1) and glypican 4 (GPC4, gene GPC4).
No difference in morphology could be seen in P0, P1, P5 or in any other passages for cells grown in the two media before or after cryopreservation. By growth curves, cell counts after thawing were similar in FBS-SM and HS-SM from days 1 to 10, with a slight trend toward higher cell counts in FBS-SM. Cells grown in both FBS-SM and HS-SM media showed similar expression of endothelial cell markers when assessed by immunohistochemistry, although the gene expression of HCEC markers was higher in HS-SM when assessed by RT-PCR: TJP1 (1,3-fold, p=0,083), ATP1A1 (2-fold, p= 0,031) and GPC4 (3-fold, p=0,064).
After cryopreservation, HS-SM was similar to FBS-SM for HCEC culture when assessed by cell morphology, proliferation, and protein expression. Marker gene expression by RT-PCR was higher in HS-SM compared to cells grown in FBS-SM.
This PDF is available to Subscribers Only