Organ Culture Medium Research Articles

Comparison of the structural integrity and quality of corneal endothelium stored in organ culture storage medium versus Eusol-C.

In this experimental study, we compared the structural integrity and cell quality of corneal endothelium stored in organ culture medium (OCS) and Eusol-C. The experiment included rabbit and human cornea experiments in vitro. Thirty rabbit corneas and thirty-two human corneas were collected and divided into two groups. All right corneas were allocated in experiment group and left corneas were placed in control group. The corneas in experimental group were stored in OCS at 34°C, and the corneas in control group were stored in Eusol-C at 4°C for 7, 14, 21, 28, and 35days, respectively. Endothelial cell morphology, cell count, and trypan blue staining for viability were assessed before storage (Day 0) and at days 7, 14, 21, 28 and 35. The structural integrity of human corneal endothelial cell was analyzed using immunohistochemistry. The samples of storage solution for microbial culture were collected on the third day and at the end of storage. The results show that no bacterial and fungal infections were found in both groups. After 14days of storage, the morphology of endothelial cell was better in the experimental group than in the control group. The endothelial cell stored in OCS were better than those stored in Eusol-C at the end of storage times, except human cornea 14days storage group. The ZO-1 protein staining showed the typical polygonal morphology of endothelial cell stored in the OCS. Corneal endothelial cells stored in the OCS had better quality up to 28days. It can be applied to Chinese eye banks as a method of corneal preservation.

Microbiological screening of corneas stored in organ culture medium at Lions Eye Bank of Western Australia from 2011 to 2022.

The purpose of this study was to analyse the contamination rate of corneal samples stored in OCM at Lions Eye Bank of Western Australia over a 12-year period. All OCM samples used to preserve corneas from 2011 to 2022 (inclusive) underwent microbiological testing. Samples were collected into aerobic and anaerobic culture bottles on day 3-5 of corneal preservation and 24h after transfer to thinning medium. Samples were tested for 7days using the BACTEC FX system. Corneas remained in quarantine until clearance was obtained. From 2011 to 2022, 3009 corneas were retrieved and 2756 corneas were stored in OCM. Thirty one (1.1%) positive samples were reported, with 20 growths of bacterial origin and 11 fungal. Microbial contamination was mostly identified on day 1 of culture (77.5%). Donors of contaminated samples had a mean age of 55years, with 17 male and 14 female donors. The highest incidence of contamination came from donors whose cause of death was cancer. Death to enucleation times of contaminated samples ranged from 3.5 to 25.5h (mean = 13.5 ± 7.3) and death to preservation time ranged from 4.1 to 27.5h (mean = 14.8 ± 7.2). These did not significantly differ from the average time from death to enucleation (mean = 13.9 ± 3) and death to preservation (mean = 16.3 ± 4.2) of non-contaminated samples. Microbiological screening of corneas stored in OCM at LEBWA showed a very low rate of positive cultures with no predictive donor characteristics.

Laboratory exploration of the use of ripasudil in descemetorhexis with a human ex vivo model

The aim of the study was to investigate the effect of ripasudil on corneal endothelial cell survival and migration after two types of descemetorhexis on a human ex vivo model. Eleven human corneoscleral buttons were incubated in either 50 ml organ culture medium containing 10 μM ripasudil or 50 μl dimethyl sulfoxide (DMSO), the vehicle in ripasudil for 2 days prior to wound creation then for 14 days after. The wound was created with either full trephination scoring or by shallow trephination plus manual peeling. At day 14, immunohistochemistry with vimentin and Na+/K+/ATPase markers was conducted. Tissues were assessed at day 3, 7 and 14 for morphology, cell migration, cell viability and cell density. Full trephination scoring created more damage on tissues compared to shallow trephination with full Descemet membrane peeling. In the full trephination scoring group, no differences in cell viability were noted when ripasudil and DMSO were compared. With the peeling method, Ripasudil could protect the endothelial cell death and maintain the morphology compared to the control. At day 14, no differences in the peripheral cell viability and density were found between ripasudil and DMSO, although the ripasudil group presented significantly increased central cell count and cell viability. Increased cell migration was noted with ripasudil and the initial cell morphology of those migrated cells was similar to that of fibroblasts. In conclusion, ex vivo modelling suggested that peeling resulted in less cell damage than scoring and ripasudil maintained better morphology and promoted migration. These effects might be via transformation of endothelial cells into a more motile spindle-like phenotype.

Descemet Membrane Endothelial Keratoplasty With and Without Graft Deswelling: A Prospective Clinical Study.

The purpose of this study was to assess whether omission of dextran from corneal organ culture medium alters the outcome of Descemet membrane endothelial keratoplasty. Participation in this single-center, multisurgeon, prospective, randomized, comparative clinical trial was offered to patients scheduled for Descemet membrane endothelial keratoplasty between April 2020 and May 2022. Patients received grafts from corneas deswollen in organ culture medium-containing 6% dextran T-500 or from corneas that were not deswollen. Corrected distance visual acuity (CDVA), graft detachment, central corneal thickness (CCT), and corneal endothelial cell counts were measured at different time points up to 12 months postoperatively. Grafts stored with dextran were transplanted in 92 patients, and grafts stored without dextran were transplanted in 102 patients. Mean donor age and endothelial cell counts did not differ significantly between both groups. Mean (±SD) postmortem time in hours was 23.9 ± 11.8 in grafts that were deswollen and 28.2 ± 13.8 in grafts that were not deswollen (P = 0.02). The groups did not show any significant difference at baseline regarding sex, CDVA, or CCT. In the group with dextran, patients had a mean age of 72.5 ± 9.9 years versus 69.5 ± 8.7 in the group without dextran (P = 0.03). CDVA improved and CCT decreased significantly in both groups. No differences were detected between the groups regarding CDVA, CCT, endothelial cell counts, or rebubbling rates. This study did not detect any evidence that the omission of dextran from organ culture medium negatively affects the outcomes of Descemet membrane endothelial keratoplasty.

Sterile donor tomography for improvement of refractive results after keratoplasty

Purpose: Any corneal microsurgeon may have inadvertently transplanted a donor corneal tissue with (subclinical) keratoconus or prior keratorefractive surgery, which typically leads to postoperative refractive surprises and cannot always be detected in the eye bank with only slit lamp. Given the increased number of keratorefractive procedures performed in the last decades, eye banks will soon have to face this problem more intensively. Thus, the efficiency and reliability of anterior segment optical coherence tomography (AS‐OCT) as a non‐invasive and sterile screening method in the eye bank to detect corneal grafts with curvature and/or thickness abnormalities was assessed.Methods: 1315 donor corneal tissues mounted in sterile tissue cultivation flasks were imaged using an AS‐OCT. The corneal tissues were preserved in organ culture medium I and transferred into organ culture medium II (containing dextran T‐500 6%) for deswelling at least 24 h prior to measurement and keratoplasty. Depth raster scans were performed through the posterior surface of the corneal tissues within a 7 mm central zone to create 3D volume data. The volume data set was preprocessed to eliminate flask reflections artefacts and to define the region of interest (ROI). Subsequently, the adaptation of a sphero‐cylindrical surface model was carried out with raytracing. Thus, the radii of curvature for the front and back surfaces can be determined and a corneal thickness map is generated.Results: The mean steep/flat front surface radius was 7.46 ± 0.29 (5.07–9.29)/7.69 ± 0.24 (6.70–9.50) mm, the corresponding values for the back surface being 6.47 ± 0.32 (5.00–8.00)/6.80 ± 0.31 (5.81–8.00) mm and the mean central thickness was 610 ± 84 (379–1457) μm. Abnormalities (beyond ±2 or ± 3 SD) were found in 140 or 34 corneas (10.6% or 2.6%) for anterior surface curvature, 120 or 38 corneas (9.1% or 2.9%) for the posterior surface, and 55 or 16 corneas (4.2% or 1.2%) for thickness.Conclusions: The AS‐OCT provides an objective, sterile and semi‐automated screening method to identify corneal morphological and refractive alterations (e.g. keratoconus, status post PRK/LASIK) and further optimize corneal donor selection in the eye bank. Corneal donors with curvature or thickness abnormalities ±3 SD do not have to be discarded but can be used for posterior lamellar keratoplasty or tectonic keratoplasty.

Preparation and outcome of swollen versus dextran deswollen grafts in descemet membrane endothelial keratoplasty (DMEK)

Aims/Purpose: To evaluate the influence of organ culture medium containing dextran on graft characteristics during preparation as well as the outcome of subsequent Descemet Membrane Endothelial Keratoplasty (DMEK).Methods: 31 corneoscleral rims of the Eye Bank of the University Hospital of Cologne with a donor age between 52 and 74 years were examined between 2018 and 2022 for their intraoperative preparation conditions and the outcome after performing 31 DMEKs. For this purpose, corneoscleral rims were randomized into two organ‐cultured groups, one immersed in culture medium with dextran (group A, n = 16) and the other in medium without dextran (group B, n = 15). The intraoperative graft preparation conditions were noted by using a standardized scoresheet, including splitting and stripping behaviour, central and peripheral attachment as well as graft fragility. The post‐operative outcome parameters were defined as Best Spectacle‐Corrected Visual Acuity (BSCVA), central and peripheral corneal thickness (CCT and PCT), endothelial cell count (ECC) and re‐bubbling rate.Results: In both groups, all 31 DM‐endothelium grafts were usable for DMEK. There was no significant difference in the intraoperative preparation conditions of swollen versus dextran deswollen DM‐endothelium grafts in respect to stripping (p = 0.722) and to Descemet membrane splitting behaviour (p = 0.013). The fragility of the grafts showed no significant difference (p = 0.433), as well as the peripheral scars indicating a pseudophakic donor (p = 1.000) and the tendency for central tearing (p = 0.484) during preparation. No significant difference in the outcome 12 months after DMEK in conditions of swollen versus dextran deswollen DM‐endothelium grafts regrading CCT (p = 0.189), re‐bubbling (p = 0.704), PCT (p = 0.063), could be observed. BSCVA was significantly better in group B 12 months after DMEK with p = 0.032 (group A 0.08 ± 0.38: group B 0.03 ± 0.13).Conclusions: This pilot study does not show relevant and significant differences between swollen versus dextran deswollen grafts in DMEK transplantation. Future prospective studies with higher patient numbers are needed. Our study suggests also non‐deswollen donors as suitable for DMEK surgery.

Preloaded DMEK with endo-in technique: Standardizing and minimizing the learning curve over 5 years using 599 corneal tissues.

To report the outcomes of standardizing pre-loaded DMEK with endothelium-inwards and its associated learning curve. Between 2017 and 2021, a total of 599 tissues were stripped using 'trephine and strip' method and loaded by folding the tissue as a taco-fold with endothelium-inwards. The folded tissues were pulled inside the funnel of a 2.2 mm IOL cartridge and stored for the desired number of days in organ culture media supplemented with dextran. Donor characteristics, endothelial cell loss (ECL) and mortality assessed by trypan blue positivity before and after stripping, and eventful cases during stripping/loading were recorded. The tissues found unsuitable for transplant after stripping (6.7%) were significantly higher compared with loading (0.67%). Central or peripheral tears, fragility of the tissues, and insufficient endothelial cell density mainly attributed towards the discard rate. Mean ECL from pre-stripping to post-stripping was 0.27% with endothelial cell mortality of 0.64% at the end of stripping. Cumulative endothelial mortality fold change (pre-strip to post-strip) was high in the first two years of operation (18.9%), which reduced to 5.1% in the following three years with significant difference (p = 0.0352). Average tissue wastage (3 operators) from first 1-150 tissues was 3%, which significantly reduced to 0.9% after achieving the learning curve (151-250) (p = 0.0492). DMEK graft preparation requires a learning curve. However, an operator with DMEK stripping skills can easily adapt to pre-loading a DMEK graft in endothelium-inwards fashion with minimal learning curve.

Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro - A Stepwise Linear Regression Algorithm.

To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm. Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO2 atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm. The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007). The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro.

The Lions Eye Bank of Western Australia: 35 Years of Eye Banking (1986–2021)

Purpose: The purpose of this study was to review the trends in preparation, storage, and usage of donor corneal tissue from 35 years of eye banking at the Lions Eye Bank of Western Australia (LEBWA). Methods: Donor and recipient records for the period July 1986 to July 2021 were evaluated with relation to donor demographics, cause of death, storage medium, corneal utilization, type of corneal graft, and indication for graft. Results: Six thousand two hundred nine eyes from 3328 donors were retrieved in the 35-year period reviewed. Sixty-six percent of eye donors were male, and 33% were female. The median age of donor was 61 years (2–98 years). The most frequent cause of donor death was a cardiac event, followed by intracerebral hemorrhage and malignancy. A total of 5219 corneal transplants were performed, with a corneal utilization rate of 84%. Storage of corneal tissue was primarily in Optisol-GS until 2010, when organ culture medium was introduced. Of 5219 recipients, 52% were male and 47% were female. The median recipient age was 63 years (1–98 years). Penetrating keratoplasty was the most common surgery performed over the 35-year period, followed by endothelial keratoplasty. The most common indications for grafts were ectasia/thinning, repeat corneal transplants, endothelial dystrophies, and postcataract surgery edema. Conclusions: We present trends in the acquisition and utilization of corneal tissue across 35 years at LEBWA. The most significant changes over this period include methods of corneal storage and advances in surgical techniques that has led to a greater usage of endothelial grafts.

Results of ultrathin Descemet stripping automated endothelial keratoplasty with donor corneas preserved in synthetic organ culture media.

To evaluate outcomes of ultrathin Descemet stripping automated endothelial keratoplasty (UT-DSAEK) with donor corneas preserved at 31°C in Cornea Syn®, a medium formulated with recombinant human serum albumin (rHSA) to replace foetal calf serum, and deswelled-transported in the xeno-free medium Cornea Trans®. Prospective, multicentre, open-label study. We evaluated the endothelial cell loss (ECL) as the percentage variation of the endothelial cell density (ECD, cells/mm2) between 6 and 12 months after surgery, corneal transparency and thickness at 12 months, and adverse events within 12 months. Endothelial lenticules of mean 89 μm, ECD ≥ 2300 cells/mm2, minimum signs of cell mortality or morphology alterations, were dissected by microkeratome in the eye bank, and grafted in patients ≥ 18 years without corneal neovascularisation, conjunctivalization, or blinking impairment. Thirty-five patients underwent UT-DSAEK, 3 showed primary failure, 1 late failure, and 2 skipped the 6-month visit. We analysed data from 29 patients, 27 with Fuchs endothelial corneal dystrophy (FECD) and 2 with pseudophakic bullous keratopathy (PBK). The median ECL between 6 and 12 months was 2.6% (p = .054, CI 0 to 12.5) and the absolute mean (SD) was 158.4 (364.1) cells/mm2. After 12 months, 96.5% of corneas were clear, with mean pachymetry of 585.9 (50.4) µm. The ECL rate after UT-DSAEK match overall that observed in DSAEK or UT-DSAEK models of endothelial survival and the overall safety compared that reported for similar follow-up. Corneas maintained in Cornea Syn® and Cornea Trans® did not affect the ECD and functional outcomes of UT-DSAEK up to 12 months.

Exploration of the ocular surface infection by SARS-CoV-2 and implications for corneal donation: An ex vivo study.

The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors. To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors. In this study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms. Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/.

Comparison of clinical outcomes after precut DMEK with or without dextran-containing medium compared to standard DMEK: a prospective pilot study.

To investigate the clinical outcome and complication rate of precut Descemet membrane endothelial keratoplasty (DMEK) in two different culture conditions, dextran-containing and dextran-free medium, and compare the results with the current standard DMEK procedure. A prospective study of 32 eyes suffering from Fuchs endothelial dystrophy were scheduled for DMEK with a follow-up of one year. The eyes were divided into four subgroups. Group + D (n = 7) received a precut DMEK stored in dextran-containing transport medium, and Group - D (n = 9) received a precut DMEK without dextran-containing medium. The respective fellow eyes received a standard DMEK (S) (preparation directly prior to surgery) stored in dextran-containing medium (S-D + ; n = 7) or without (S-D-; n = 9). Clinical outcome (visual acuity, endothelial cell count, central corneal thickness) and rebubbling rate were comparable for all four groups. None of the patients had a graft failure. The preliminary data of the pilot study show that precut liquid-bubble DMEK leads to comparable clinical results regardless of dextran-containing or dextran-free organ culture medium and is further comparable to the standard DMEK procedure.

333 Poor perifollicular vascularization is associated with nutrient insufficiency and a quiescent metabolic phenotype in intermediate hair follicles from patients with female pattern hair loss

Female pattern hair loss (FPHL) is a non-scarring form of alopecia, resulting from premature catagen induction leading to telogen effluvium and progressive transformation of terminal (t) scalp hair follicles (HFs) into intermediate/miniaturized (i/m) HFs. Currently available treatment strategies, are often combined with nutritional supplements. However, data supporting nutrient deficiency in FPHL HFs are lacking. Therefore, we attempted to investigate this by asking whether any changes can be seen in perifollicular vascularization in FPHL and by examining HF nutritional status and metabolism in affected and non-affected HFs. CD31 quantification revealed significantly reduced numbers of perifollicular blood vessels in the relatively unaffected (occipital) but most prominently in the affected (parietal) scalp from FPHL patients compared to occipital scalp from healthy donors. This was accompanied by changes in the interfollicular expression of angiogenesis- associated growth factors VEGF and thrombospondin-1. Metabolic analyses showed that nutrients important for adequate hair growth such as biotin, pantothenic acid, L-cysteine, and L-glutamic acid were decreased in iHFs compared to tHFs in affected FPHL scalp skin. iHFs also displayed a more quiescent metabolic phenotype, characterized by a lower rate of aerobic glycolysis and glutaminolysis during organ culture. Yet, lesional t or i/m FPHL HFs were capable of taking up labelled exogenous nutrients when these were added in excess into the organ culture medium. Taken together, our data suggest that t and i/m HFs in FPHL affected scalp skin are poorly vascularized which likely compromises nutrient delivery to the HFs and that nutrient supplementation may be capable in principle to correct this deficiency.

NEONATAL OVARIAN RESERVE FOLLOWING CHEMOTHERAPY EXPOSURE, EX VIVO MURINE MODEL

To determine the impact of exposure to maternal serum chemotherapy on formation of the ovarian reserve. Breast cancer complicates 1:3000 pregnancies, and current safety data is limited to congenital anomalies and early childhood outcomes. Chemotherapy is known to have significant germ cell toxicity, and yet little has been done to understand the transgenerational effect of in utero exposure. Murine ovarian reserve establishment mimics the human fetus but with a significant time shift to postnatal development. A randomized ex-vivo animal study with 100 postnatal day 0 C57BL/6 mouse ovaries was performed under IACUC approval. After sacrifice, sister ovaries were randomized to control (drug carrier alone) or drug exposure. Planned exposures were derived from widely used chemotherapy treatment regimens for maternal malignancy: doxorubicin, cyclophosphamide, paclitaxel, docetaxel, and cisplatin. Ovaries were cultured in hanging well organ culture media with addition of exposure on culture day 2. Drug dosing replicated known Cmax concentration. A mid dose of half the Cmax was also used to gain greater understanding of dynamics. A single drug exposure was utilized, and planned analysis occurred at 48 hours and 5 days. Immunofluorescence with TRA98 and VASA was used to quantify oocyte number and density. Data was then transferred to Graphpad Prism to generate descriptive statistics and apply a two-way ANOVA for each condition. Under control conditions, oocytes matured in culture with a moderate fall in density as oocyte size increased. Exposure to doxorubicin results in a loss of 95.2% of the oocyte density seen in the control at 7 days. At 7 days control ovaries have an oocyte density 570.3 oocytes/mm2, but Cmax exposure of doxorubicin results in a decrease to 27.2 oocytes/mm2. A two-way ANOVA confirms the significance of this relationship (p<0.0001). Exposure to cyclophosphamide resulted in less of a decrement. At 7 days the oocyte density decreased by 50.5% compared to the control to 282.2 oocytes/ mm2. Two-way ANOVA again confirmed significance (p=0.0004). Cisplatin, docetaxel and paclitaxel all demonstrated unique phenotypical changes on the ovaries and their oocytes, but none resulted in a significant decrease in oocyte density. The unique exposure of in utero chemotherapy may result in profound premature ovarian insufficiency. Our ex-vivo murine model suggests that maternal chemotherapy choices might be made to craft a less harmful chemotherapy regimen to ensure fetal health. By avoiding doxorubicin and cyclophosphamide, the delicate establishment of ovarian reserve in the fetus can continue even as the mother pursues life-saving chemotherapy treatment. Further work in vivo and in follow-up of human cohorts is needed to confirm these findings.

Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin β4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2–9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.

P–624 Doxorubicin exposure detrimentally effects establishment of early ovarian reserve, ex-vivo mouse model

Abstract Study question Does exposure to chemotherapy agents used to treat cancer during pregnancy cause significant decrease in early establishment of the ovarian reserve? Summary answer Significant oocyte loss is noted following exposure of early mouse ovaries to doxorubicin. This apoptotic decline is not seen with cisplatin, docetaxel or paclitaxel exposure. What is known already Chemotherapy has been shown to adversely affect the ovarian reserve of pre-pubertal and reproductive age females. However, little is known regarding the effects in the next generation following treatment in pregnancy beyond observational population studies that examined rates of birth defects and developmental delays. Reproductive function in offspring develops over time and would be difficult to quantify in these short-term studies. An ex-vivo mouse ovary culture offers the ability to closely mimic maternal treatment level serum concentrations and directly evaluate the impact on oocyte number and cell death markers. Study design, size, duration In mice, the ovarian reserve is matured postnatally, mimicking the biologic second trimester activity of the human ovary. Wild-type C57bl/6 pups were collected on postnatal day 0. Ovaries were cultured in hanging well organ culture media with addition of DMSO or a chemotherapy agent. Immunofluorescence was used to quantify oocyte number and density. Power calculation showed a N of 11 per drug would be needed to demonstrate a decrease of ⅔ or more. Participants/materials, setting, methods: 83 ovaries were cultured, sectioned and analyzed in duplicate. Planned analysis at serum max and mid concentrations was performed at 48 hours and 5 days. Given clinically variation in concentrations of cisplatin, additional concentration samples were added. After noting the degradation following exposure to doxorubicin, additional samples at earlier time points of 12 and 24 hours of exposure were added for dynamic evaluation. Means were calculated and then compared using a 2-way ANOVA. Main results and the role of chance Doxorubicin exposure during establishment of the ovarian reserve resulted in a significant loss of oocyte number and density. At 12 hours a 22% decrease was noted; this increased to a loss of 91% of oocytes by 48 hours of exposure. The oocyte density fell from 693 oocytes/ mm² in the control to only 63 oocytes/ mm² in the serum max concentration (p = 0.003). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) revealed early impact within the stroma by 12 hours with widespread apoptotic changes by 24 hours. Treatment with cisplatin resulted in a phenotypic change in the oocyte population with preservation of smaller, more peripheral cells. The average oocyte density remained similar to control even at the highest clinical concentrations, 536 oocytes/ mm² compared to 570 oocytes/ mm² (p = 0.772). Docetaxel and paclitaxel demonstrated an increase in oocyte number and density, though not enough to reach significance. The oocyte density 5 days following docetaxel exposure was 802 oocytes/ mm², a 41% increase (p = 0.12). The oocyte density 5 days following paclitaxel exposure was 780 oocytes/ mm², a 37% increase (p = 0.817). The drug exposure did impact stromal cells, as noted in TUNEL images. Limitations, reasons for caution This ex-vivo mouse model offers tight control of chemotherapy concentration, it does not account for filtration and modification by the placenta. Longer term cultures may also demonstrate that temporary arrest in small oocytes, such as those exposed to cisplatin, do not thrive and later progress to apoptosis. Wider implications of the findings: Doxorubicin is employed as the most common chemotherapy during pregnancy. Utilization may be to the serious detriment of the younger generation. If other alternatives are clinically effective they should be considered. No other models have explored the effect of fetal ovary exposure to chemotherapy on the establishment of ovarian reserve. Trial registration number Not applicable

Time- and Stimulus-Dependent Characteristics of Innate Immune Cells in Organ-Cultured Human Corneal Tissue

Purpose: The pattern of immune cells infiltrating the corneal stroma has been extensively studied in mice, but data on human tissue have been far less elaborate. To further characterize the number and differentiation state of resident immune cells in organ-cultured human corneal tissue, we employed a comprehensive bioinformatic deconvolution (xCell) of bulk RNA-sequencing (RNA-seq) data, immunohistochemistry (IHC), and flow cytometry (FC). Methods: A transcriptome-based analysis of immune cell types in human corneal samples was performed. The results were validated by IHC, focusing on the identification of pro-inflammatory (M1) and regulatory (M2) macrophages. A protocol was established to identify these 2 different macrophage populations in human corneal tissue by means of FC. Subsequently, corneal samples in organ culture were differentially stimulated by IL-10, IL-4 & IL-13, or LPS and macrophage populations were evaluated regarding their response to these stimuli. Furthermore, cell survival was analyzed in correlation with time in organ culture. Results: xCell-based mathematical deconvolution of bulk RNA-seq data revealed the presence of CD8 T cells, Th17 cells, dendritic cells, and macrophages as the predominant immune cell types in organ-cultured human corneal tissue. Furthermore, RNA-seq allowed the detection of different macrophage marker genes in corneal samples, including PTPRC (CD45), ITGAM (CD11b), CD14, and CD74. Our RNA-seq data showed no evidence of a relevant presence of monocytes in human corneal tissue. The presence of different macrophage subtypes was confirmed by IHC. The disintegration and subsequent FC analysis of human corneal samples showed the presence of both M1 (HLA-DR⁺, CD282⁺, CD86⁺, and CD284⁺) and M2 (CD163⁺ and CD206⁺) macrophage subtypes. Furthermore, we found that the total number of macrophages in corneal samples decreased more than the total cell count with increasing tissue culture time. Treatment with IL-10 led to higher total cell counts per cornea and to an increased expression of the M2 marker CD163 (p < 0.05) while expression levels of various M1 macrophage markers were not significantly reduced by interleukin treatment. Conclusions: Regarding different macrophage populations, untreated human corneas showed more M1 than M2 macrophages. With increasing organ culture time, these macrophages decreased. In terms of cell dynamics, adding interleukins to the organ culture medium influenced the phenotype of macrophages within the cornea as detected by FC. Modifying the immunomodulatory properties of human grafts appears a promising approach to further reduce the risk of graft rejection in patients. In this context, treatment with interleukins was more effective in upregulating M2 macrophages than in suppressing M1 macrophages in corneal tissue.

Epithelial Regeneration in Human Corneas Preserved in an Active Storage Machine.

PurposeTo characterize the corneal epithelium (CE) and limbal epithelium (LE) of human corneas stored in an innovative active storage machine (ASM) after a period of organ culture (OC).MethodsCorneas unsuitable for graft and stored in a standard commercial OC medium for 2 to 5 weeks were transferred into our ASM for 14 days. The ASM actively maintained an overpressure on the endothelial side (20 mm Hg) while ensuring medium renewal. We compared three modalities of storage in the ASM's epithelial chamber: (1) alternating exposure to a supplemental hormonal epithelial medium (SHEM) and air (air-lifting), (2) continuous immersion in SHEM, and (3) continuous immersion in OC medium. Passive immersion of the whole cornea in OC medium or of the CE in SHEM with or without airlifting served as controls. Paired corneas were used for better comparability. Histology, differentiation (by immunolabeling), and ultrastructure were analyzed at the end.ResultsThe ASM with air-lifting was most effective in regenerating a pluristratified and differentiated CE (apical ZO-1 and MUC16 staining and regeneration of the glycocalyx). In addition, the LE was stratified with preserved expression of ABCB5. The ASM with immersion in SHEM or OC medium gave a less stratified and differentiated CE. In the three control groups, the epithelia, when present, were paucistratified and less differentiated.ConclusionsIn human corneas previously stored in OC, the ASM regenerates a CE with differentiation characteristics close to normal.Translational RelevanceRegeneration of the epithelium of human corneas discarded by eye banks will increase tissue availability for research.

A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due to the lack of causative therapies, there is an urgent need for innovative experimental organ culture models to study the mechanisms involved in the progression of the disease, find therapeutic targets, and reduce the need for animal models. We here present a novel, three-dimensional organ culture model protocol mimicking the proinflammatory and catabolic microenvironment, which is present during IDD. Initially, bovine caudal IVDs were dissected, cleaned, and cultured in the tissue culture medium. Dynamic physiologic or pathologic loading was applied in a custom-made bioreactor for 2 hours per day. IVDs were assigned to a control group (high glucose medium, physiological loading, phosphate-buffered saline injection) and a pathological group (low glucose medium, pathological loading, tumor necrosis factor-alpha injection) for four days. Gene expression analysis from collected nucleus pulposus cells of the IVDs and enzyme-linked immunosorbent assay of the conditioned organ culture media was performed. Our data revealed a higher expression of inflammatory markers and reduced disc heights after loading in the pathological group compared to the control group. This protocol is reliable to simulate IVD inflammation and degeneration and can be further expanded to broaden its application scope.

Thickness and Curvature Changes of Human Corneal Grafts in Dextran-Containing Organ Culture Medium Before Keratoplasty.

To determine the changes of corneal thickness and curvature of human corneal grafts in organ culture medium II, containing dextran T500 6%, before keratoplasty. We examined the tomography of 24 corneas from our eye bank transferred from medium I into medium II. Images were repeated hourly during 24 hours using an anterior segment optical coherence tomography. The central corneal thickness (CCT) was measured with the manual measurement tool of the anterior segment optical coherence tomography. The radii of curvature (anterior flat and steep and posterior flat and steep) were measured with a MATLAB self-programmed software for "sterile donor tomography." The mean CCT (±SD) at baseline (T0) was 727 ± 156 μm. It reached 581 ± 103, 506 ± 84, 472 ± 79, and 456±7 μm after 6, 12, 18, and 24 hours, respectively. After 12 hours, 83% of the final deswelling was achieved. The radii of curvature (±SD) at baseline (T0) were (posterior flat, posterior steep, anterior flat, and anterior steep) 6.6 ± 0.5, 6.2 ± 0.5, 7.7 ± 0.4, and 7.4 ± 0.4 mm, respectively. After 24 hours, the radii of curvature reached 6.8 ± 0.1, 6.6 ± 0.3, 7.6 ± 0.1, and 7.4 ± 0.2 mm, respectively. The kinetics of the deswelling process in medium II follow a hyperbolic curve. Considering a CCT of 506 μm at T12, we assume that a time interval of 12 hours in medium II might be enough for clinical purposes. This result might help to keep storage in medium II as short as possible to escape potential toxic effects of dextran in medium II. The radius of curvature does not seem to change within 24 hours for all measured surfaces.