Organ Culture Storage Research Articles

Donor cornea transfer from Optisol GS to organ culture storage: a two‐step procedure to increase donor tissue lifespan

PurposeStorage time for donor corneas in Optisol GS is limited compared to Eye Bank Organ Culture (EBOC). We here examine the epithelium on donor corneoscleral rims after primary storage in Optisol GS and subsequent incubation in EBOC.MethodsMorphology was monitored by light and electron microscopy, expression of phenotypic and genotypic markers by immunohistochemistry and RT-PCR and changes in oxidative lipid and DNA damage by ELISA and COMET assay.ResultsA prominent loss of cells was observed after storage in Optisol GS. After maintenance in EBOC, spreading apical cells were Occludin+, while the staining for E-cadherin and Connexin-43 was less intense. There were an upregulation of Occludin and a downregulation of E-cadherin and Connexin-43. Eye Bank Organ Culture was associated with an ongoing proliferative activity and a downregulation of putative progenitor/stem cell marker ABCG2 and p63. Staining for 8-OHdG and Caspase-3 did not increase, while levels of malondialdehyde and number of DNA strand breaks and oxidized bases increased.ConclusionsThis dual procedure should be pursued as an option to increase the storage time and the pool of available donor corneas. The observed downregulation of markers associated with stemness during EBOC is relevant considering the potential use of donor epithelium in the treatment of ocular surface disorders.

Endothelial Cell Density Before and After the Preparation of Corneal Lamellae for Descemet Membrane Endothelial Keratoplasty With a Stromal Rim

To assess the endothelial cell density before and after the preparation of precut posterior corneal lamellae for Descemet membrane (DM) endothelial keratoplasty with a stromal rim with the aim of standardizing this new technique and establishing the parameters of corneas intended for manual preparation under the conditions of an ocular tissue bank. Two groups of corneoscleral buttons were used to prepare lamellae consisting of a central zone of endothelium-DM surrounded by a supporting peripheral stromal rim. Group 1 contained 12 corneas with a live endothelial cell density (LECD) of 2500 cells per square millimeter or more. Group 2 contained 10 corneas with a density of 2500 cells per square millimeter or less. The preparation was performed manually using an artificial anterior chamber. The endothelial cell density (ECD) and the percentage of dead cells were assessed before and immediately after preparation and after 48 hours of organ culture storage at 31°C. Immediately after preparation, on average, 4.9% and 5.2% of dead cells were found in group 1 and group 2, respectively. After 48 hours of storage, the percentage decreased significantly in both the groups. There was no significant decrease in the ECD 48 hours after preparation in group 1; however, there was a significant decrease in group 2. The amount of tissue wasted during preparation was 24%. The decrease in ECD is dependent on the initial values: the cell loss is less in corneas with higher original densities. We suggest that the minimal acceptable LECD of lamellae should be 2500 cells per square millimeter.

A Decrease in the Density of HLA-DR-Positive Cells Occurs Faster in Corneas Stored in Organ Culture than under Hypothermic Conditions

Aims: To compare the number of antigen-presenting cells (APC) at various locations in fresh human corneoscleral disks and in those that are stored for grafting under hypothermic conditions or in organ culture (OC) with the aim of determining the conditions under which the decline in APC numbers is most substantial. Methods: Cryosections obtained from fresh corneoscleral disks and disks stored under hypothermic (Optisol-GS) or OC conditions were used. The density of HLA-DR-positive cells was determined on cross sections using enzyme immunohistochemistry (alkaline phosphatase-antialkaline phosphatase technique). Longitudinal sections were used for detecting ATPase activity. Results: The densities of HLA-DR-positive cells in both the epithelium and stroma increased from the central (3.79 and 0.61/mm²) to the peripheral cornea (5.56 and 1.35/mm²) as well as to the limbus and conjunctiva. A marked decrease in the number of HLA-DR-positive cells occurred after 7 days of storage in all corneal areas, the limbus and conjunctiva, compared to fresh tissue. No positive cells were found in the epithelium of corneas after 14 days in OC and after 21 days in hypothermic storage. Twenty-eight days of storage in OC led to the complete absence of HLA-DR-positive cells in the epithelium of the limbus and conjunctiva, and to a significant reduction in the stroma. Conclusion: Corneas stored in OC longer than 14 days are likely to be less immunogenic than corneas stored under hypothermic conditions, thus resulting in a possible positive effect on prolonging graft survival.

Pre‐cut posterior corneal lamellae with a stromal rim used for Descemet’s membrane endothelial keratoplasty with a stromal rim (DMEK‐S)

Abstract Purpose To standardize a technique for the manual preparation of posterior corneal lamellae for DMEK‐S under the conditions of an ocular tissue bank. To assess the qualitative and quantitative parameters of the lamellar endothelium with the aim of establishing the parameters of corneas intended for preparation. Methods Corneoscleral buttons 17mm in diameter not suitable for grafting were used. Group 1 contained 12 corneas with a live endothelial cell density ≥ 2500 cells/mm2. Group 2 contained 10 corneas with a density lower than 2500 cells/mm2. The lamellae, consisting of a central 6mm zone of endothelium‐Descemet’s membrane surrounded by a supporting peripheral stromal rim, were prepared manually using the big bubble technique. The percentage of dead cells and the live endothelial cell density were assessed before and immediately after preparation as well as after 48 hours of organ culture storage using light microscopy following 0.15% trypan blue and 0.9% sucrose treatment. Results Immediately after the preparation, on average 4.9 % and 5.2 % of dead cells were found in group 1 and group 2, respectively. The percentage of dead cells decreased significantly during subsequent storage in organ culture in both groups (to 1.5 % and 1.7 % for group 1 and 2). There was no significant decrease in live endothelial cell density in group 1 during 48 hours of culturing; however, there was a significant decrease in group 2. Conclusion The decrease in endothelial cell density during organ culturing depends on the original values. We suggest that the minimal acceptable live endothelial cell density of corneas intended for preparation should be 2500 cells/mm2 to ensure the best clinical outcomes.

Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas

Aims: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture. Methods: Customized electrodes without endothelial contact were developed. Two plasmids containing the cytomegalovirus promoter and reporter genes [enhanced green fluorescent protein (eGFP) or beta-galactosidase (β-gal)] were electroporated in 2 series of human corneas with eight 1-Hz 100-ms pulses of 125 mA square current. Controls were exposed to naked DNA without electric pulses. eGFP-transduced corneas were used to determine the transgene expression kinetics, whereas β-gal measured transfection efficiency using image analysis tools. Overall, endothelial toxicity was determined by: (1) cytotoxicity tests using triple staining with Hoechst 33342, ethidium homodimer III, and calcein AM, 3 h and 3 and 14 days after electroporation on the series of 15 eGFP-transfected paired corneas; (2) anti-ZO-1 staining to assess tight junctions’ integrity. Results: All electroporated corneas carried transfected ECs, whereas the controls carried none. eGFP expression was observed 3 h after electrotransfer, and was then present from days 1 to 28. Transfection efficiency determined on 63 corneas transfected with β-gal ranged from 0.1 to 54% of the transfected ECs (mean ± SD: 7 ± 11%, median: 2.9%) with significant reproducibility for paired corneas from the same donor. Electroporation produced low early EC death. Anti ZO-1 staining revealed no dramatic change in EC mosaic continuity, neither 1 and 3 nor 28 days after electroporation. Conclusions: Gene electrotransfer to the endothelium of organ-cultured human corneas with custom-designed electrodes allows rapid and easy EC transfection. However, further optimization is required to ensure reproducible results.

Gene therapy: can we prevent/modulate apoptosis in EC?

Abstract Purpose Regardless of the inciting cause, CEC loss is a common denominator of corneal graft failure. CEC loss during storage results in significant loss of suitable tissue for grafting, CEC loss after transplantation is a major cause of graft failure. The purpose of this study is to investigate the role of apoptosis in CEC in order to prevent CEC loss during storage. Methods Gene transfer of Lenti‐Bcl‐xL or –p35 was accomplished in human donor corneas, primary cultured CEC and an immortalized CEC line and compared to untreated controls. Cell death (apoptosis) was induced by Actinomycin or Etoposide (external vs. internal apoptotic pathway, respectively). In addition, CEC loss during preservation was studied both during Optisol GS (4C) and organ culture storage (37C, Biochrome Medium I). Both storage media were diluted with PBS to promote cell loss. CEC were enumerated, apoptosis was detected by TUNEL staining and confocal microscopy. Results The percentage of TUNEL‐positive CEC provoked by the apoptotic inducers was significantly reduced relative to controls. Transfected corneas preserved an almost intact endothelial monolayer while controls nearly entirely lost vital CEC. During long‐term storage experiments at 4C and at 37C, CEC counts in corneas expressing anti‐apoptotic genes remained significantly higher compared to the controls. Conclusion Protection of CEC by anti‐apoptotic genes appears to be an effective method to reduce CEC loss during storage. The application of this technique could increase the amount of high quality grafts in eye banking and further reduce graft failure following corneal transplantation, and is be of specific interest as to precut corneas and DSAEK procedures.

Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damage

To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions. The central endothelium of 12 pairs of human corneas with similar endothelial parameters was damaged to create a 1 mm(2) lesion. One cornea from each pair was stored under OC and one under hypothermic conditions. The endothelial cell density (ECD), coefficient of variation, hexagonality and percentage of dead cells were assessed before and after damage and on days 7, 14, 21 and 28 of storage. The mean ECD of corneas subsequently stored under OC or hypothermic conditions was 2764/mm(2). Immediately after damage, a denuded Descemet's membrane with a few remaining dead cells was observed at the injured area. After 7 days of storage under OC conditions, almost no dead cells were observed at the place of injury. A non-significant worsening of the qualitative parameters (polymegatism and pleomorphism) was found. After 14 days, ECD was 1933/mm(2) and 2478/mm(2) centrally and pericentrally, respectively. Similar values were found after 21 and 28 days of storage. The lesions with remnant dead cells persisted throughout hypothermic preservation. From day 14 the corneas became cloudy and in poor condition, while the pericentral ECD was 2523/mm(2). The reparative capacity of the cornea is maintained under OC but not under hypothermic conditions. For corneas containing dead endothelial cells, OC is therefore the method of choice because it may improve the quality of the stored tissue.

Corneal oedema after cataract surgery: predisposing factors and corneal graft outcome

Pseudophakic bullous keratopathy (PBK) is one of the main indications for corneal transplantation. Graft survival and visual outcome in this group are often poorer than for other indications. The aim of this study was to find risk factors for developing corneal oedema after cataract surgery and factors that influence the subsequent survival of the graft and the visual outcome. We carried out an observational, retrospective cohort study using data from the Swedish Cornea Transplant Register and patient medical records. A total of 273 patients whose indication for corneal transplantation was corneal oedema after cataract surgery were included in the study. Multiple logistic regression analysis and, where appropriate, univariate analyses were applied. A total of 43% of the patients developed persistent corneal oedema immediately after cataract surgery, the main risk factors for which were phacoemulsification and pre-existing endothelial disease. Almost a third (32%) of the transplants for PBK failed within 2 years, for which rejection and other postoperative complications increased the risk. Half (50%) the patients had visual acuity < or = 0.1 at 2 years after keratoplasty. Comorbidity, increasing duration of the bullous keratopathy and increasing age affected the visual outcome negatively. Phacoemulsification was a risk factor for immediate persistent corneal oedema after cataract surgery, although it did not increase the overall risk of developing PBK. However, transplants for immediate PBK had a better survival rate than those for later onset PBK. Shorter duration of PBK and intraocular lens exchange at the time of penetrating keratoplasty increased the likelihood of good visual acuity.

Regeneration With Proliferation of the Endothelium of Cultured Human Donor Corneas With Extended Postmortem Time

To examine the endothelium of donor corneas with extended postmortem time for survival and reparative mechanisms in an eye bank organ culture storage system. We obtained 14 pairs of donor corneas with a postmortem time ranging from 29 to 163 hours. One cornea of a pair was immediately fixed for the study of structural changes postmortem and to serve as a control. The second was stored in organ culture for 3 days and thereafter fixed to be studied for reparative processes. Examination was done with light microscopy and scanning electron microscopy. Immunohistochemical staining with antibodies against proliferating cell nuclear antigen, Ki-67, and n-cadherin was performed to examine for cell proliferation and to characterize the cells. The control corneas showed increasing endothelial cell damage with increasing postmortem time. After 5-7 days postmortem, most cells were structurally damaged. After 3 days in organ culture, all corneas acquired an endothelial covering of the posterior surface, with cells, suggesting proliferation in both scanning preparations and in cross-sections. Positive endothelial cell staining with proliferating cell nuclear antigen was found in all cultured corneas. Ki-67 staining of the endothelium was found in 9 of the cultured corneas. The study showed survival of the corneal endothelium up to 7 days postmortem, and accordingly, the potential clinical use of donor corneas with extended postmortem time. Our results furthermore suggest that repair of the endothelium in donor corneas during organ culture storage occurs also by proliferation and not only by migration and enlargement of existing cells. If we uncover the mechanisms regulating cell proliferation in corneal endothelium, it should be possible to develop better storage methods of corneal transplants to improve quality and supply.

Effects of Organ Culture and Optisol-GS Storage on Structural Integrity, Phenotypes, and Apoptosis in Cultured Corneal Epithelium

A previous report has described the use of eye bank storage of cultured human limbal epithelial cells (HLECs) to provide a reliable source of tissue for treating limbal stem cell deficiency. In the present study, conventional organ culture (OC) storage and Optisol-GS (Bausch & Lomb, Irvine, CA) storage of cultured HLECs were compared. Three-week HLEC cultures were either organ cultured at 31 degrees C or 23 degrees C or stored in Optisol-GS at 5 degrees C in a closed container for 1 week. Morphology was studied by light microscopy and transmission electron microscopy, and phenotypic characterization was assessed by immunohistochemistry. Apoptosis was evaluated by real-time RT-PCR microarray analysis, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). The ultrastructure was preserved at 23 degrees C, while storage at 31 degrees C and 5 degrees C was associated with enlarged intercellular spaces, separation of desmosomes, and detachment of epithelial cells. Cultured HLECs remained undifferentiated in all storage conditions. The expression of the antiapoptotic gene BCL2 was prominently upregulated in storage at 23 degrees C and 5 degrees C. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 in 23 degrees C and 5 degrees C storage conditions suggests a reduction in nuclear factor-kappaB activity. No significant increase in cleaved caspase-3 and TUNEL staining was observed in response to eye bank storage, and the labeling indices of cleaved caspase-3 (range, 0.0%-4.7%) and TUNEL (range, 0.0%-7.8%) were low. These data indicate that OC storage of cultured HLECs at ambient temperature is superior to OC storage at 31 degrees C and Optisol-GS storage at 5 degrees C and that apoptosis is minimal after eye bank storage of cultured HLECs.

Frequency of Positive Donor Rim Cultures After Penetrating Keratoplasty Using Hypothermic and Organ-Cultured Donor Corneas

To compare the frequency of positive rim cultures after penetrating keratoplasty using corneas preserved by hypothermic and organ culture storage. To evaluate the influence of standard procurement techniques on the frequency of microbial donor rim contamination. Six hundred four donor corneas stored at 31 degrees C and 214 at 4 degrees C were studied. Microbiology studies were carried out during organ culture storage, and corneas with positive medium cultures were discarded. Frequency of postoperative positive rim cultures was related to the type of corneal storage and procurement technique used. Thirty-nine (6.4%) corneas with positive medium cultures were discarded during organ culture. Microbiology reports of 628 donor rims cultures from 671 (94%) consecutive transplants were reviewed. Positive rim cultures resulted in 24 (3.8%) cases. None of the patients developed endophthalmitis. The frequency of postoperative positive rim cultures was greater after hypothermic than organ culture storage, being 9.8% and 1.3%, respectively (chi(2) = 24.9; P < 0.001). With organ culture storage, the frequency of positive rim cultures was 1.3% and 1.4% after enucleation and in situ corneal excision, respectively (chi(2) = 0.03; P = 0.638). After hypothermic storage, positive rim cultures were found in 8% of the corneas procured using enucleation and 12% of the corneas excised in situ (chi(2) = 0.829; P = 0.254). Organ culture storage allows one to recognize and discard corneas with microbial contamination during storage. This method significantly reduces the frequency of postoperative positive rim cultures compared with hypothermic storage. Procurement methods do not influence the percentage of positive rim cultures.

Regeneration of the epithelium in organ‐cultured donor corneas with extended post‐mortem time

The maximum post-mortem time limit for obtaining donor corneas varies between eye banks. It is not known for how long a time the epithelial cells survive post-mortem, nor is it known if donor corneas with extended post-mortem time are able to regenerate the epithelium. Therefore, we wanted to examine the epithelium in donor corneas for regenerative ability during storage in an eye bank organ culture system. Twenty-four paired donor corneas with post-mortem time from 28 to 163 hr were obtained. One cornea of a pair was fixed immediately to serve as a control, and the second was cultured in eye bank medium at 32 degrees C for 3 days. Examination of the specimens was performed with light and scanning electron microscopy. Immunohistochemical staining methods with antibodies against K 3, K 19, vimentin and p63 were used to further characterize the cells. The control corneas showed decreasing amounts of epithelial cells with increasing post-mortem time. All the cultured corneas demonstrated rapid regeneration of the epithelium. After 3 days in organ culture, 10 of 12 donor corneas were covered with epithelium. Even up to 7 days post-mortem, viable cells reside in the corneal epithelium. The study demonstrates the hardiness and enormous regenerative potential of peripheral corneal cells. Donor corneas processed in an eye bank organ culture storage system will obtain an intact epithelial layer within a few days.

Major Endothelial Loss From Corneas in Organ Culture

The aim of this study was to show that major losses can still occur on corneas judged suitable for grafting at the first count. In addition, we studied the frequency of these losses on 1992 corneas over a period of 4 years to evaluate the risk incurred. We evaluated the incidence of these major losses and the associated risk factors. An Ishigawa diagram was created with the Cornea Bank team and the ophthalmologists involved in organ retrieval. Endothelial losses caused by bacterial or fungicidal contamination were excluded from the study. For the 29 corneas that suffered major losses, we analyzed the donor files for donor age, clinical file, geographical origins of the corneas, the person who did the retrieval, the length of time the cornea was stored, the data resulting from examining the endothelium at the bank by optical microscope, and the method used for sterilizing the material used. Specific analyses in cases of major loss of endothelial content: anatomopathologic examination of the corneas and search for the herpes simplex virus (HSV; type 1 or 2) by polymerase chain reaction (PCR). We carried out a statistical analysis using a chi(2) test on the 1992 corneas studied to see if the presence of diabetes (type 1 or 2) in the donor led to reduction levels different from those of corneas originating from nondiabetic donors. The incidence was evaluated at between 0.4% and 3% of corneas sampled, and the associated risk factor was between 0.8% and 6% of grafted corneas. The occurrence of major losses was independent of donor age and was independent of the person who did the retrieval. The occurrence of major losses was independent of geographical origin. We tested our media for endotoxin before use and found levels from 0.22 to 3.9 UI/mL. We verified the absence of a chronological relationship between the batches of media used in the bank and the number of major losses observed, showing that the pyrogenicity limit was independent of cytotoxicity limits. Data analysis showed no difference in reduction levels between diabetic and nondiabetic donors (P < 0.05). Results on the detection of HSV-1 by PCR on the storage media were all negative, and these results agree with the anatomopathologic examinations that showed no signs of viral infection. Total endothelial losses amounted to 1.4%/yr. Without the double endothelial counts, we would have had 29 primary graft rejections over that period. During storage, this loss has not been linked to a specific cause, but risk factors such as traumatic death, herpes infections, and badly controlled endotoxin levels should be considered when taking preventative actions. For the moment, a second endothelial count before grafting should be carried out, because all these problem grafts conformed to grafting criteria after the first count. The possibility of carrying out this second count is one of the recognized advantages of storage in organ culture.

Donor corneas for transplantation: a scanning electron microscopic study of the epithelium

Donor corneas are processed in eye banks and used for transplantation as a standard routine. The maximum time limit post-mortem for harvesting donor tissue varies greatly between eye banks. This study aimed to examine the corneal epithelium for structural changes post-mortem. A total of 51 corneas harvested between 14 and 163 hours post-mortem were examined using scanning electron and light microscopy. Cell loss occurred through desquamation of flat superficial cells during the first days. In corneas with a post-mortem time of more than 2-3 days, large superficial cell sheets and deeper cells detached, starting centrally. Deep peripheral cells remained. The loss of the superficial cells revealed the 3-dimensional structure of the epithelium and the membrane characteristics of deeper cells. The longer the time post-mortem, the greater the epithelial cell loss. However, a rim of peripheral cells remained, even after 7 days. The superficial cell layer showed signs of strong lateral attachment and broke up in a sheet-like fashion. The intercellular adhesion between deeper cells and adhesion between the basal cells and the basement membrane appeared to be weak post-mortem. The cell membrane structures of the remaining cells were surprisingly well retained. The clinical implication of the study is discussed.

Experimental Study of the Survival of Metastatic Cancer Cells in Corneal Organ Culture

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. 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. 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. 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.

Corneal Epithelial Cultures Generated from Organ-Cultured Limbal Tissue: Factors Influencing Epithelial Cell Growth

Purpose: To explore the in vitro proliferative potential of human limbal epithelial cells after 31°C organ-culture storage and to investigate putative factors influencing it. Methods: 185 cultures of limbal explants were carried-out either from full-thickness explants (n = 102) or from enzymatically dissociated cells (n = 83) seeded on a feeder layer of human keratocytes. Epithelial outgrowth was assessed by phase contrast microscopy using a computerized image analysis software. Cell phenotype was evaluated by transmission electron microscopy and immunocytology. Univariate and multivariate analysis were performed to determine factors influencing epithelial growth in culture. Results: An epithelial outgrowth of 100 square mm or more was observed in 52% of cultures, (average growth area: 440 +/− 256 mm at three weeks). Corneal epithelial phenotype was confirmed by transmission electron microscopy, and cytokeratin pattern. Cytokeratine 19, deltaNp63, nestin and vimentin positive staining revealed undifferenciated epithelial cells in both explant and cell suspension cultures at three weeks. Short death to cornea retrieval time (p < 0.03) and female donors (p < 0.01) were associated with higher cell growth. Enzymatic treatment of explants by trypsin, but not dispase, decreased cell proliferation at two (p < 0.03) and three weeks (p < 0.04). Donor age, duration of corneal storage, and source of the explant did not influence the cell growth. Conclusion: Organ-culture conditions can preserve limbal cell mitotic potential if limbal tissue is excised early after circulatory arrest. Human keratocytes can be used as a feeder layer allowing epithelial cells to maintain poorly differentiated phenotype in culture. Further investigations are needed to explain the influence of the donor sex on epithelial cell growth in culture.

Animal Compound–Free Medium and Poloxamer for Human Corneal Organ Culture and Deswelling

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. 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. 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. 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.

MMP-type endopeptidase activity in the cornea. Its evolution during organ culture storage at the Eye Bank. Effect of hyaluronan

About 46% of total corneas obtained from donors in the French Eye Bank cannot be grafted for several reasons as loss of endothelium or other. Corneal cells express proteolytic enzymes, essentially matrix metallo-proteinase MMP-2 and MMP-9. In presence of hyaluronan and some other GAG-s their activity increases as could be shown on keratocyte cultures. Hyaluronan concentration increases during in vitro preservation and can represent a serious hasard for corneal conservation. The control of MMP-release and activation might well be one of the factors involved in graft deterioration. We could show however that only a slight fraction (≤12%) of total, relatively high endopeptidase activity of the cornea is released in the media during storing. It appears therefore that most of the proteolytic activity determined in corneal extracts remains confined to the stroma and might not represent an important risk for preservation, at least for the endothelium.

Endotoxin in storage medium of human corneal grafts and clinical course after penetrating normal-risk keratoplasty.

It is well known that endotoxins in storage medium may stimulate cytokine production and expression of adhesion molecules as well as endothelial damage in human corneal grafts. It has been supposed that endotoxin exposure of corneal grafts may, therefore, cause immune reactions and lead to reduced endothelial cell count after penetrating keratoplasty. It was the purpose of this prospective study to evaluate if this hypothesis is true. A consecutive series of 274 samples of sterile organ culture storage medium from 274 human corneal grafts was collected between August 1998 and February 1999 and tested for endotoxin using Limulus amebocyte-lysate assay (LAL) after 7 days of organ culture. Threshold endotoxin level was set at 1.0 U/ml. A total of 161 grafts were transplanted and 113 were discarded. Within the 161 corneas transplanted, 62 were grafted to normal-risk patients and 99 to high-risk patients. Only normal-risk keratoplasty patients were included in the study and followed for at least 10 months. Immune reactions, graft failures, and postoperative endothelial cell counts were recorded. The mean endotoxin level in organ culture medium of all transplanted grafts was 1.07+/-2.96. Mean endotoxin level in organ culture medium of discarded grafts was 1.68+/-5.76, with 71 samples being below and 42 above the threshold of 1.0 U/ml called endotoxin-negative and endotoxin-positive, respectively. In all 36 culture medium samples from the 62 grafts transplanted to the group of normal-risk keratoplasty patients were endotoxin-negative and 26 endotoxin-positive. An influence of endotoxin levels on incidence of immune reactions, graft failure, and postoperative endothelial cell counts could not be revealed in patients with normal-risk keratoplasty. Low endotoxin levels in storage medium neither seem to promote immune reactions nor to contribute to postoperative chronic endothelial cell loss in normal-risk keratoplasty patients.

Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium.

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. 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. 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. 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.