P21-A141†Quantification of bioactive factors in human serum eyedrops.

PURPOSE: NHS Blood and Transplant supply serum eye drops (SED) for the treatment of severe dry eye syndrome, however, understanding of what components of SED contribute to their activity is limited. SEDs are produced from a patient's own blood or from an allogeneic donor source. The serum component is separated from the whole blood which is then diluted 50/50 with sterile saline, and contains bioactive molecules that are believed to help heal and maintain the ocular surface. The objective of this study is to quantify the amount of bioactive molecules in donor serum, and to understand how processing variables effects these factors. METHODS: Samples of SEDs from 28 male allogenic donors were taken from ultra-low temperature storage and thawed. They were then centrifuged at 13,000 rpm at 4oC to remove potential contaminants such as residual red blood cells. Duplicate test samples were analysed for epidermal growth factor (EGF) and fibroblast growth factor (FGF) using ELISA kits. Analysis was carried out using Excel. RESULTS: The age range of the donors was 17 to 79 years (mean 47.9).Mean time from venepuncture to refrigerated storage was 6 hours 12 minutes with time ranging from 2 hours 40 minutes to 9 hours 35 minutes.The concentration of EGF found in the diluted serum ranged from 0.048 to 1.90 ng/ml (mean 0.87 ng/ml), and FGF concentration ranged from 4.88 to 39.50 pg/ml (mean 12.37 pg/ml).Analysis showed that there was no correlation between either age of the donor, or sample transfer time and growth factor concentration. CONCLUSION: Our study demonstrated that with both types of growth factors measured in the SED, a wide range of concentrations were found in the donor samples. Compared to published data EGF was at higher range while FGF was lower. Further analysis of other factors present in the donor serum is being undertaken to determine if any pattern can be found.

14†Validation of transport systems to ensure quality of tissue delivered to eye banks.

INTRODUCTION: NHS Blood and Transplant Tissue and Eye Services (TES) retrieve eyes for corneal and scleral transplant purposes from hospitals, hospices, and funeral homes throughout the UK. The eyes are sent to TES eye banks either in Liverpool or Bristol. A major objective of TES is to ensure that the eyes arrive at their destinations in good condition and remain fit for purpose. With that in mind TES Research and Development have conducted a series of validation studies to ensure that eyes are packaged appropriately, and that the material is not damaged and maintains the required temperature during transport. Whole eyes are shipped on wet ice. MATERIALS AND METHODS: Whole eyes - a corrugated plastic carton with an expanded polystyrene insert (Ocular Correx) had been used by Manchester and Bristol eye banks for at least 15 years before the eye banks joined TES. This original transport carton was compared with a re-useable Blood Porter 4 transport carton consisting of a single expanded polystyrene base and lid with a fabric outer packing. Porcine eyes were used secured in eye stands. T-class thermocouple probes were inserted through the lid s of 60 ml eye pots via pre-drilled holes, with the probe touching the outer surface of the eye, with probes routed under the boxes lid. For the original carton, three different weights of wet ice (1, 1.5, and 2 kg) were used inside the box, with the box placed in an incubator (Sanyo MCO-17AIC) at 37oC. Thermocouples were also placed in the wet ice and the incubator itself before they were connected to a calibrated datalogger (Comark N2014) which recorded the temperature every 5 minutes. For the Blood Porter carton a single 1.3 kg weight of ice was used RESULTS: Whole eyes - tissue temperature was maintained between 2-8oC for 17.8 hours with 1 kg wet ice, 22.4 hours with 1.5 kg wet ice and 24+ hours with 2 kg wet ice. With the Blood Porter 4 box tissue temperature was maintained between 2-8oC for more than 25 hours with 1.3 kg wet ice. DISCUSSION: Data reported in this study showed that both types of box are able to maintain tissue temperature between 2-8oC for at least 24 hours, provided the correct amount of wet ice is used. The data also showed that tissue temperature did not drop below 2oC, meaning there was no danger of the cornea potentially freezing.

3†Emergency salvage of time expired clinical corneas during the COVID-19 pandemic.

INTRODUCTION: Corneas for clinical use can be stored for a maximum of 28 days in organ culture medium after death. At the beginning of the COVID-19 pandemic in 2020 it became apparent that; a rare situation was arising in that clinical operations were being cancelled and that there would be a surplus of "clinical grade" corneas. Consequently, when the corneas reached the end of the storage period, if the tissue had appropriate consent, they were transferred to the Research Tissue Bank (RTB). However, University research had also stopped due to the pandemic and there was a situation where the RTB had good quality tissue without any users. Rather than discarding the tissue, a decision was made to store the tissue for future use by cryopreservation. MATERIALS AND METHODS: An established protocol for cryopreserving heart valves was adapted. Individual corneas were placed into wax histology cassettes then inside a Hemofreeze heart valve cryopreservation bag with 100 ml cryopreservation medium (10% Dimethyl sulphoxide)). They were frozen in a controlled rate freezer (Planer, UK) to below -150oC and stored in vapour phase over liquid nitrogen (VPLN) below -190oC. To assess morphology, six corneas were cut in half, one half was processed for histology whilst the other half was cryopreserved, stored for 1 week then thawed and processed for histology. The stains used were Haematoxylin and Eosin (H&E) and Miller's with Elastic Van Gieson (EVG). RESULTS: Comparative histological examination indicated that there were no visible, major, detrimental changes in morphology in the cryopreserved group as compared to the controls. Subsequently, a further, 144 corneas were cryopreserved. Samples were assessed for handling properties by eye bank technicians and ophthalmologists. The eye bank technicians felt that the corneas may be suitable for training purposes such a DSAEK or DMEK. The ophthalmologists said that they had no preference between the fresh or cryopreserved corneas, and both would be equally suitable for training purposes. CONCLUSION: Time expired, organ-cultured corneas, can be successfully cryopreserved using an established protocol by adapting the storage container and conditions. These corneas are suitable for training purposes and may prevent discard of corneas in future.

Correction: Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone.

[This corrects the article DOI: 10.1371/journal.pone.0177416.].

Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone.

To improve the safe use of allograft bone, decellularization techniques may be utilized to produce acellular scaffolds. Such scaffolds should retain their innate biological and biomechanical capacity and support mesenchymal stem cell (MSC) osteogenic differentiation. However, as allograft bone is derived from a wide age-range, this study aimed to determine whether donor age impacts on the ability an osteoinductive, acellular scaffold produced from human bone to promote the osteogenic differentiation of bone marrow MSCs (BM-MSC). BM-MSCs from young and old donors were seeded on acellular bone cubes from young and old donors undergoing osteoarthritis related hip surgery. All combinations resulted in increased osteogenic gene expression, and alkaline phosphatase (ALP) enzyme activity, however BM-MSCs cultured on old donor bone displayed the largest increases. BM-MSCs cultured in old donor bone conditioned media also displayed higher osteogenic gene expression and ALP activity than those exposed to young donor bone conditioned media. ELISA and Luminex analysis of conditioned media demonstrated similar levels of bioactive factors between age groups; however, IGF binding protein 1 (IGFBP1) concentration was significantly higher in young donor samples. Additionally, structural analysis of old donor bone indicated an increased porosity compared to young donor bone. These results demonstrate the ability of a decellularized scaffold produced from young and old donors to support osteogenic differentiation of cells from young and old donors. Significantly, the older donor bone produced greater osteogenic differentiation which may be related to reduced IGFBP1 bioavailability and increased porosity, potentially explaining the excellent clinical results seen with the use of allograft from aged donors.

Polyurethane scaffolds seeded with CD34(+) cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors.

We describe a 3D erythroid culture system that utilises a porous polyurethane (PU) scaffold to mimic the compartmentalisation found in the bone marrow. PU scaffolds seeded with peripheral blood CD34(+) cells exhibit a remarkable reproducibility of egress, with an increased output when directly compared to human bone scaffolds over 28 days. Immunofluorescence demonstrated the persistence of CD34(+) cells within the scaffolds for the entirety of the culture. To characterise scaffold outputs, we designed a flow cytometry panel that utilises surface marker expression observed in standard 2D erythroid and megakaryocyte cultures. This showed that the egress population is comprised of haematopoietic progenitor cells (CD36(+)GPA(-/low)). Control cultures conducted in parallel but in the absence of a scaffold were also generally maintained for the longevity of the culture albeit with a higher level of cell death. The harvested scaffold egress can also be expanded and differentiated to the reticulocyte stage. In summary, PU scaffolds can behave as a subtractive compartmentalised culture system retaining and allowing maintenance of the seeded "CD34(+) cell" population despite this population decreasing in amount as the culture progresses, whilst also facilitating egress of increasingly differentiated cells.

Development of an improved bone washing and demineralisation process to produce large demineralised human cancellous bone sponges.

Shaped demineralised bone matrices (DBM) made from cancellous bone have important uses in orthopaedic and dental procedures, where the properties of the material allow its insertion into confined defects, therefore acting as a void filler and scaffold onto which new bone can form. The sponges are often small in size, <1.0 cm(3). In this study, we report on an improved bone washing and demineralisation process that allows production of larger DBM sponges (3.375 or 8.0 cm(3)) from deceased donor bone. These sponges were taken through a series of warm water washes, some with sonication, centrifugation, 100 % ethanol and two decontamination chemical washes and optimally demineralised using 0.5 N hydrochloric acid under vacuum. Demineralisation was confirmed by quantitative measurement of calcium and qualitatively by compression. Protein and DNA removal was also determined. The DBM sponges were freeze dried before terminal sterilisation with a target dose of 25 kGy gamma irradiation whilst frozen. Samples of the sponges were examined histologically for calcium, collagen and the presence of cells. The data indicated lack of cells, absence of bone marrow and a maximum of 1.5 % residual calcium.

Optimized demineralization of human cancellous bone by application of a vacuum.

Human demineralized bone matrix derived from cortical bone is used by surgeons due to its ability to promote bone formation. There is also a need for shaped demineralized bone matrices made from cancellous bone, where the properties of the material allow its insertion into defects, therefore acting as a void filler and scaffold onto which new bone can form. In this study, we report that demineralized bone sponges were prepared by dissecting and cutting knee bone into cancellous bone cubes of 1 cm(3) . These cubes were then taken through a series of warm water washes, some with sonication, centrifugation, and two decontamination chemical washes. The cubes were optimally demineralized into sponges with 0.5N hydrochloric acid under vacuum with constant pH measurement. Demineralization was confirmed by quantitative measurement of calcium and qualitatively by compression. The sponges were freeze dried before terminal sterilisation with a target dose of 25 kGy gamma radiation whilst frozen. Samples of the sponges were histologically examined for calcium and collagen and also tested for osteoinductivity. Data showed well defined collagen staining in the sponges, with little residual calcium. Sponges from two out of three donors demonstrated osteoinductivity when implanted into the muscle of an athymic mouse.