Experimental study of the survival of metastatic cancer cells in corneal organ culture.

PURPOSE: Transmission of donor malignancy to the recipient could be one of the most disastrous complications of corneal grafting. Because of the scarcity of donor tissue and the lack of sufficient scientific evidence, the harvest of donor tissues from deaths due to systemic malignancy is permitted. This study was conducted to investigate the possible transmission of donor metastatic disease via corneal tissue preserved in organ culture (OC) conditions. METHODS: The viability of four frequent human cancer cell lines (lung, breast, skin, and colon) was studied in OC. Various inoculums of cancer cells labeled with the membrane marker PKH67 were seeded on donor corneas and preserved in OC, followed by cell-tracking studies, histopathology, and immunohistochemistry. HLA matching of the dissected Descemet's membrane (DM) of preserved corneas was conducted, to demonstrate cell adherence. Primary cell culture was performed to confirm the viability of adherent tumor cells. RESULTS: Viability tests showed a poor but persistent survival of cancer cells after 2 weeks in OC. Cell tracking, histopathology, and immunohistochemistry demonstrated cancer cell adherence to donor endothelium. HLA typing of the DM of preserved corneas revealed the presence of cancer cell alleles. Primary culture of the DM showed cell proliferation that was identical with the original cancer cell line, according to HLA studies. CONCLUSIONS: The findings demonstrate that under laboratory conditions, metastatic cancer cells adhere to donor corneal tissue, survive, and retain proliferative capacity during storage in OC. Cell lines differ in their viability potential, as well as the pattern of adherence to donor endothelium. Further in vivo experimentation in laboratory animals is need to determine the safety of such harvests.

Animal compound-free medium and poloxamer for human corneal organ culture and deswelling.

PURPOSE: Eliminating fetal calf serum (FCS) from corneal organ culture (OC) media has long been a challenge. This study was an assessment of a new animal compound-free (ACF) medium for corneal storage and of its combination with poloxamer for end-of-storage corneal deswelling. METHODS: A randomized controlled study with masked assessment compared the ACF medium to standard commercialized media containing 2% FCS and their combination with dextran for deswelling. Paired human corneas were randomly allocated at procurement, one to the ACF medium and the other to the FCS media, and then assessed at day (D)2 and D30 of OC storage and after 48 hours of deswelling. Comparison criteria were endothelial cell density (ECD) and morphometry by a corneal analyser, quality of endothelial visualization (using saline), EC mortality (trypan blue), corneal thickness, corneal transparency, and folding. Fifty-six corneas (28 pairs) with ECD of 2000 cells/mm(2) or more were enrolled. Data were compared using paired tests with P < 0.01 deemed significant. RESULTS: Parameters were similar at baseline (D2) between groups. Daily EC loss during the 30 days of storage was reduced with the ACF compared with standard (-0.31% +/- 0.30% vs. -0.88% +/- 0.38%, P < 0.001). With poloxamer 188 (Lutrol F68; BASF, Ludwigshafen, Germany), EC loss was substantially reduced (-1.43% +/- 3.60 vs. -15.41% +/- 10.13%, P < 0.001) and morphometry better preserved, despite thickness reduction, transparency improvement and folding reduction comparable to dextran. After 30 days of storage in ACF medium and deswelling in poloxamer 188, ECD was 30% higher (2466 +/- 447 cells/mm(2) vs. 1729 +/- 281 cells/mm(2), P < 0.001). ACF medium alone and combined with poloxamer 188 considerably facilitated EC visualization at D30 and after deswelling. CONCLUSIONS: The ACF medium combined with poloxamer 188 for deswelling showed superiority over standard FCS medium in its ability to preserve EC viability and facilitate endothelial visualization. This innovative use of poloxamer for deswelling appears far less toxic than does dextran.

Urgent need for normalization of corneal graft quality controls in French eye banks.

BACKGROUND: Assessment of corneal tissue quality before graft is mainly based upon the determination of endothelial cell density (ECD) by eye banks. These cells are responsible for corneal transparency, and ECD correlates with graft survival. In France and often elsewhere in Europe, ECD is measured using a "naked-eye" procedure under a light microscope. To measure objectively the reliability of ECD determination in France, we developed four test corneas with a known ECD. METHODS: The test corneas consisted of 1 mm2 of human corneal endothelium with stained cell borders. The 64 technicians of the 21 French eye banks counted according to the protocol applied in their respective centers. RESULTS: More than half of the 256 counts (152, 59%) deviated by more than 10% from actual ECD. Of the counts, 85 (33%) were over-estimated, and 67 (26%) were under-estimated. Deviation ranged between 42% under-estimation and 82% over-estimation. Eight banks (38%) constantly over-estimated, and nine (43%) under-estimated ECD. Half of the inter-technician gaps within an eye bank were more than 10%, with a maximum of 51%. CONCLUSIONS: This audit highlights the unacceptable lack of reliability of manual ECD assessment in French eye banks. This surely indicates the delivery of poor quality corneas for graft in certain centers and wastage in others. We urgently advocate normalization of French counting methods. This may require upgrading to a computer-aided method.