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Diseases and injuries that compromise the ocular surface cause considerable patient distress and have long term consequences for their quality of life. Treatment modalities that can address the delicate balance of tissue regeneration, inflammation and maintenance of corneal transparency are therefore needed. We have recently formulated two novel eye drops from placental tissues: cord blood platelet lysate (CBED) and amniotic membrane extract eye drops (AMED), which can be used to treat severe ocular disorders. Here we characterise these two preparations by measuring: (a) growth factors (GF) and cytokines composition, (b) promotion of human corneal epithelial cell (HCEC) growth and (c) effects on immune cells in a lymphocyte culture assay. Finally, their bioavailability was assayed in an ex vivo porcine corneal model. We show that both preparations contain GF and cytokines that were able to promote the in vitro growth of HCEC and support repair in an in vitro scratch test. When assessed in a lymphocyte culture, both favoured immune suppression reducing the cellular expression of NKG2D and CD107a as well as the production of interferon gamma (IFN-γ) in natural killer, NKT and T cells. Regarding bioavailability, CBED active molecules were found mainly in the pre-corneal fraction with some penetration into the corneal fraction, in an ex vivo model. In summary, both placental-derived allogeneic preparations, CBED and AMED, display regenerative and immunomodulatory capabilities. These results will help define mechanisms of action and the best indications and doses of each product for use in a particular patient and support the development of off-the-shelf therapies for ocular surface pathologies in which wound healing defects and inflammatory events are contributing factors.

Cord blood platelet rich plasma (CB-PRP) derivatives have been investigated as potential therapeutic agents for the treatment of diverse conditions including ocular surface disease and skin ulcers. We have developed processes for the formulation of several CB-PRP preparations, which have different composition and attributes. Here we describe the molecular characteristics of these preparations and we make recommendations as to their most appropriate clinical application based on functional and immunomodulatory profiles. We show that incubation of adult peripheral blood mononuclear cells (PBMCs) with all three preparations dramatically reduced the production of INFγ and the expression of NKG2D and CD107a in NK, NKT, and T cells thus diminishing their activation, we propose that the likely mechanism is the high levels of soluble NKG2D ligands present in plasma. Of the three preparations we investigated, CB platelet lysate (PL) and platelet releaseate (PR) have higher concentrations of trophic and pro-angiogenic factors, CB platelet poor plasma (PPP) has the lowest concentration of all analytes measured. Based on these finding we propose that CB-PR is the most suitable raw material for skin wound patches, while CB-PL and PPP can be used to prepare eye drops for severe ocular surface pathologies and inflammatory conditions such as corneal ulcers or severe dry eye disease, respectively.

Abstract Introduction In current public cord blood (CB) banking, many of the collected units are considered not suitable for hematopoietic stem cell transplant (HSCT) due to their low stem cell counts. New clinical applications are foreseen like specialized hemotherapy shown in the setting of wound healing (Samarkanova, 2020) or neonatal transfusion (Teofili, 2023). In this regard, CB platelet concentrates (PRP), platelet poor plasma (PPP) and red blood cells (RBC) have been used but no standard procedures for their fractionation in a single processing have been well defined. Objectives The aim of this study is to validate a processing method for obtaining all blood components in a way for a new model, the multicomponent CB banking, to expand the use of CB beyond transplantation. Methods A protocol for multicomponent CB fractionation, using commercially available, closed system was adapted at Barcelona CBB. After double centrifugation, the whole CB, is separated into three fractions: PRP, PPP and RBC. Then, CB-RBC is further diluted with saline-adenine-glucose (SAG) mannitol solution (2:1 rate) and leuco-reduced using a specific filter. Storage condition for CB-PRP and CB-PPP is <-65ºC up to 3 years and for CB- RBC at 2-6°C during 14 days. For validation, quality control testing was done, using a blood analyzer for platelet and RBC counts. Additionally, for CB-RBC additional parameters like potassium and hemolysis were assessed following EDQM guidelines. Results Ten samples underwent validation protocol achieving acceptance criteria defined. Results of validation are summarized in Table 1 Discussion CB units can be fractionated into three clinical-grade blood components using a single processing method within CB bank. In addition for CB-RBC, stability was established during two weeks on refrigerated storage. The platelet/plasma fractions are being used for topical applications as eye drops for severe ocular surface pathologies or as gels for chronic cutaneous ulcers with positive results (Samarkanova et al. 2020, 2021). Our study group is currently conducting a clinical trial to evaluate safety and efficacy of CB-RBC transfusion in very preterm infants (NCT05612919). The Multicomponent CBB is feasible and might be a useful way to expand CB applications beyond transplantation, maximizing the use of all donations. Table 1: CB derived products acceptance criteria and validation outcome (n=10): Product (sample) Parameter Acceptance criteria Results mean (range) Whole cord blood Hours >48 26 (18-34) Volume (mL) >50 mL 65 (45-88) Platelets (x10^9/L) >150 218 (153-298) Leucocytes (x10^9/L) informative 11.5 (7.5-16.9) Erythrocytes x10^12/L informative 3 (2.6-3.6) Hemolysis (%) 0 0 CB-PRP Volume (mL) >5 8.6 (5.0-20.0) Platelet count (x10^9/L) 800-1200 1046 (847-1185) CB-PPP Volume (mL) informative 46 (30-56) Platelet count (x10^9/L) informative 15.8 (8-43) CB-RBC (day 0) Volume (mL) >15 28 (18.5-37.5) Hematocrit (%) 50-70 55 (50-57%) Hemoglobin (g/dL) informative 17.5 (16.5-18.2) Erythrocyte (x10^9/L) informative 4.8 (4.4-5.2) Hemolysis (%) 0 0.05 (0-0.2) Potassium (mmol/L) informative 3.5 (1.5-6.2) CB-RBC (day 14) Hemolysis (%) <0.8 0.25 (0-0.69) Potassium (mmol/L) informative 17.2 (14.6-21.0)

Abstract Introduction The Barcelona Cord Blood Bank (BCBB) was established 28 years ago and developed the Concordia CB donation program, with over 60 maternity units in six regions of Spain and Andorra. These units are connected to a single processing and storage facility. Initially, collections were based on a total nucleated cell (TNC) count > 900x10^6. This criterion evolved, and since 2015, a strict minimum TNC of 1500x10^6 has been applied. Objectives to analyze the characteristics of the stored Cord Blood Units (CBUs) over the years to classify the cryopreserved units according to different variables, allowing for an understanding of the bank's quality and future orientation towards new needs and applications Methods a retrospective analysis to determine the percentage of high-quality units available for transplantation. For this purpose, CBUs were classified into four categories based on initial cellular quality: category 1 (>1500x10^6 TNC), category 2 (1200-1500x10^6 TNC), category 3 (900-1200x10^6 TNC), and category 4 (<900x10^6 TNC). Additionally, the geographical origin of the donors was analyzed Results Among 124,988 donations collected, 24,661 were cryopreserved (20%), of which 2,171 were canceled due to freezing problems or not meeting quality criteria and nearly 2,100 were shipped for transplantation. Currently, there are 19,094 CBUs (15% of all collected) active in the bone marrow donor registry. According cell number, CBU inventory is divided into categories, and 6,922 (36%) belong to category 1. In contrast, majority of CBU shipped comes from this category (77%), Figure 1). Interestingly, all participating regions maintained equivalent shipping rates. In terms of geographical origins, of the 19,094 cryopreserved units, 54,00% belonged to the European Community (EC), 3,95% were Latin America, 2,08% Sub-Saharan 0,07% were Asian and 39,90% did not have the data to classify the samples (Fig 2) Discussion This analysis enhances the understanding of the characteristics and quality of units requested for transplantation and the geographical distribution of donors. The current operational inventory is smaller than expected, highlighting the need to continue collecting high-quality units from diverse ethnic backgrounds to meet patient needs. A multi-regional donation model and collaboration with health professionals and social awareness are crucial for sustaining cord blood banks