We evaluated the small molecules (AFM) caffeine, curcumin and pirfenidone to find non-toxic concentrations reducing the transformation of activated human corneal stromal keratocytes (aCSK) to scar-inducing myofibroblasts (MYO-SF). CSK were isolated from 16 human corneas unsuitable for transplantation and expanded for three passages in control medium (0.5% FBS). Then, aCSK were exposed to concentrations of caffeine of 0–500 μM, curcumin of 0–200 μM, pirfenidone of 0–2.2 nM and the profibrotic cytokine TGF-β1 (10 ng/mL) for 48 h. Alterations in viability and gene expression were evaluated by cell viability staining (FDA/PI), real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. We found that all AFMs reduced cell counts at high concentrations. The highest concentrations with no toxic effect were 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone. The addition of TGF-β1 to the control medium effectively transformed aCSK into myofibroblasts (MYO-SF), indicated by a 10-fold increase in α-smooth muscle actin (SMA) expression, a 39% decrease in lumican (LUM) expression and a 98% decrease in ALDH3A1 expression (p < 0.001). The concentrations of 100 µM of caffeine, 20/50 µM of curcumin and 1.1 nM of pirfenidone each significantly reduced SMA expression under TGF-β1 stimulation (p ≤ 0.024). LUM and ALDH3A1 expression remained low under TGF-β1 stimulation, independently of AFM supplementation. Immunocytochemistry showed that 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone reduce the conversion rate of aCSK to SMA⁺ MYO-SF. In conclusion, in aCSK, 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone significantly reduced SMA expression and MYO-SF conversion under TGF-β1 stimulation, with no influence on cell counts. However, the AFMs were unable to protect aCSK from characteristic marker loss.

Human amniotic membrane (hAM) is usually stored at −80 °C. However, in many regions, cryopreservation at −80 °C is not feasible, making hAM unavailable. Therefore, the possibility of cryopreservation at −28 °C (household freezer) was investigated. hAMs (n = 8) were stored at −80 °C or −28 °C for a mean time of 8.2 months. hAM thickness, epithelial integrity and basement membrane were assessed histologically. The collagen content, concentration of hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) were determined. Elastic modulus and tensile strength were measured. The mean thickness of hAM stored at −28 °C was 33.1 ± 21.6 µm (range 9.7–74.9); thickness at −80 °C was 30.8 ± 14.7 µm (range 13.1–50.7; p = 0.72). Mean collagen content, epithelial cell number and integrity score showed no significant difference between samples stored at −28 °C or −80 °C. Basement membrane proteins were well preserved in both groups. Mean tensile strength and elastic modulus were not significantly different. Concentration of bFGF at −28 °C was 1063.2 ± 680.3 pg/g (range 369.2–2534.2), and 1312.1 ± 778.2 pg/g (range 496.2–2442.7) at −80 °C (p = 0.11). HGF was 5322.0 ± 2729.3 pg/g (range 603.3–9149.8) at −28 °C, and 11338.5 ± 6121.8 pg/g (range 4143.5 to 19806.7) at −80 °C (p = 0.02). No microbiological contamination was detected in any sample. The cryopreservation of hAM at −28 °C has no overt disadvantages compared to −80 °C; the essential characteristics of hAM are preserved. This temperature could be used in an alternative storage method whenever storage at −80 °C is unavailable.

We evaluated the suitability of 2% human platelet lysate medium (2%HPL) as a replacement for 2% fetal bovine serum medium (2%FBS) for the xeno-free organ culture of human donor corneas. A total of 32 corneas from 16 human donors were cultured in 2%FBS for 3 days (TP1), then evaluated using phase contrast microscopy (endothelial cell density (ECD) and cell morphology). Following an additional 25-day culture period (TP2) in either 2%FBS or 2%HPL, the pairs were again compared using microscopy; then stroma and Descemet membrane/endothelium (DmE) were processed for next generation sequencing (NGS). At TP2 the ECD was higher in the 2%HPL group (2179 ± 288 cells/mm²) compared to 2%FBS (2113 ± 331 cells/mm²; p = 0.03), and endothelial cell loss was lower (ECL HPL = −0.7% vs. FBS = −3.8%; p = 0.01). There were no significant differences in cell morphology between TP1 and 2, or between 2%HPL and 2%FBS. NGS showed the differential expression of 1644 genes in endothelial cells and 217 genes in stromal cells. It was found that 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (HMOX1, SERPINE1, ANGPTL4, LEFTY2, GADD45B, PLIN2, PTX3, GFRA1/2), and the downregulation of pro-inflammatory/apoptotic genes (e.g., CXCL14, SIK1B, PLK5, PPP2R3B, FABP5, MAL, GATA3). 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and producing potentially beneficial alterations in gene expression.

Concerns of possible transmission of SARS-CoV-2 from donors to patients by corneal transplantation have caused a decline in corneal transplantations. Graft culture media are routinely tested for infectious risks, but it is unclear whether this constitutes a viable means to avoid transmitting SARS-CoV-2 via keratoplasty. We found that SARS-CoV-2 RNA was not present in the medium after seven days of organ culture of corneas from donors (n = 4), who were SARS-CoV-2-positive upon tissue procurement. These medium samples showed no presence of viral RNA. To pursue this question under controlled conditions and further exclude the possibility of productive infection in corneal grafts, we inoculated corneoscleral discs from healthy donors (n = 8) with infectious SARS-CoV-2 and performed PCR testing of the culture medium at various time points. After seven days of culture, we also tested for SARS-CoV-2 RNA within the inoculated corneal tissue. The medium from tissue samples inoculated with SARS-CoV-2 showed no increase in viral RNA, which may indicate lack of viral replication in these corneal grafts. SARS-CoV-2-RNA was, however, detected on or in corneal tissue seven days after inoculation. Our data suggest that corneal grafts may not be permissive for replication of SARS-CoV-2 and demonstrates that PCR testing of culture media cannot safely exclude that tissue has been exposed to SARS-CoV-2. It also demonstrates the difficulty to differentiate between virus adherence and virus replication by PCR testing in SARS-CoV-2 exposed tissue.

Cornea transplants are tissue transplants and, as such, must be distinguished from organ transplants (e.g. heart or kidney transplants). However, tissue transplants can only be performed if there are enough donors available to attend to patients in need. Unfortunately, there are too few organ and tissue donors in Germany. All steps involved in processing donor tissues must be performed in accordance with the highest quality standards. All tasks and measures are aimed at improving patient care in the surgical units that are to be supplied. Cornea banks are subject to complex requirements, whose implementation is essential in terms of both infrastructure and personnel. The analysis and identification of essential topics reveal central fields of action that are decisive for implementing the challenges facing cornea banks. Questions of employee qualification, strategic questions due to new transplantation techniques, and changes in the societal perception of organ and tissue donation require the development of strategies that should have a holistic and sustainable effect. Received: 27 January 2021 Accepted: 15 March 2021 Publication Date: 22 June 2021 (online) © 2021. Thieme. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany