Resumen de Plasma ric en plaquetes de sang de cordó umbilical per a teràpia i desenvolupament d'assajos clínics per a tractament d'úlceres del peu diabètic y oculars
There are many advantages to using cord blood (CB) as a source of therapeutic platelet and plasma derivatives for regenerative medicine. These include availability, universal use, young donor source, and virally safe biological material, rich in tissue regenerative factors. CB 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 The aim of this thesis is to validate a bioprocess design for the production of cord blood-derived platelet concentrates (CBPC) in a public CB Bank (BST method). CBPC was defined as a product of 10±5 mL, 1,000±200×10E9/L total platelets, free of erythrocytes and leukocytes. A total of 300 CB units were centrifuged in two steps to enrich for platelets, in compliance with GMP. The samples were tested for the degree of platelet activation present, and the levels of growth factor were analyzed to evaluate their function. CBPC were then activated after thawing with 10% calcium gluconate to generate platelet gels (CBPG) to treat patients with diabetic foot ulcers. Additionally, the molecular characteristics for trophic, angiogenic factors and cytokines of different preparations derived from CB-PRP (platelet poor plasma: CB-PPP; platelet lysate: CB-PL; platelet releasate: CB-PR) were assessed to evaluate their most appropriate clinical application based on functional and immunomodulatory profiles. Finally, a case study evaluating 46 eyes unresponsive to conventional treatments and requiring urgent interventions, who received allogeneic eye drops obtained from CB-PL (CBED) to treat severe ocular surface lesions under compassionate use was performed A total of 84% of the processed CB units fulfilled the acceptance criteria. Final products contained 1,017±149×10E9 platelets/L in 10±3mL of plasma. Platelet recovery was 50±9%. BST method ensures depletion of white and red blood cells. Platelets showed low levels of activation during processing, but were significantly activated after thawing, further test shows that product are rich in growth factors. For clinical evaluation, a total of 21 CBPG were applied in 3 patients, with no reported adverse events and improvement of ulcers in all of them. Further was investigated the immunomodulatory function of CB-PRP derivatives, for this was used adult PBMCs with CB-PRP preparations that dramatically reduced the activation of NK, NKT and T cells. Of the three preparations CB-PRP that were investigated, CB-PL and CB-PR have higher concentrations of trophic (EGF, bFGF, HGF, TGF-β1) and pro-angiogenic (VEGF, PDGF AB/BB, MMP-2,9, TIMP1-4) factors, CB-PPP has the lowest concentration of all measured analytes. Based on these findings CB-PR seems the most suitable starting material for skin wound patches, while CB-PL and PPP could be used to prepare eye drops for severe corneal pathologies and inflammatory conditions such as ulcers or sever dry eye disease, respectively. Additionally, we evaluated clinical application in compassionate treatment on patients with ocular surface disorders in groups with corneal ulcers (neurotrophic, trauma, burns) which showed full and partial ulcer recovery in 25 (78%) and 6 (19%) eyes respectively. One eye (3%) did not respond to treatment. For groups with chronic conditions (dry eye and oGVHD) improvement was reported in 12 (85%) eyes, and lesions worsened on treatment in both eyes (15%) of one patient. No severe adverse events were directly attributed to CBED BST method for CBPC production is valid and reproducible, and CB-PRP derivatives are rich in trophic and angiogenic factors; with immunosuppression capacity. Promptly available CBPG and CBED resulted in a well-tolerated allogeneic therapy that showed evidence of safety and efficacy in treated patients. These positive results support final steps of clinical scale-up of both CBPG and CBED as a novel medicinal products of CB Banks.
