Mesenchymal stem cells (MSCs) have been at the centre of worldwide scientific interest for several years: both because of their incredible therapeutic potential and because of the limited ethical issues involved in their use. In recent decades it has been shown that the regenerative therapeutic efficacy of MSCs is largely due to the secretion of a pool of paracrine factors, called secretome, composed of free and soluble bioactive molecules including cytokines, chemokines and growth factors, and non-soluble extracellular vesicles (EVs) of nano or micrometric size. The secretome seems, therefore, to represent a valid alternative to cell therapy, as it offers several advantages including lower immunogenicity, greater applicability in clinical practice, good shelf life and affordable cost. Even in the veterinary field, although only recently, the therapeutic use of MSC secretome to replace parental cells has been proposed in the clinic, especially for musculoskeletal disorders (MSD) which have a high prevalence. However, it should be considered that the use of secretome in both human and veterinary clinical practice is still hampered by the lack of scalable Good Manufacturing Practice (GMP) compliant isolation processes, as well as the lack of secretome formulation in a medicinal product. In this regard, this thesis concerned the optimization of the technological-pharmaceutical development of lyophilized secretome (lyo-secretome), isolated from equine MSC from adipose tissue, the definition of its pharmaceutical quality and the evaluation of its safety and efficacy in vitro for potential use in the treatment of MSD. By combining ultrafiltration and freeze-drying, it has been possible to obtain a dry product based on equine MSC secretome, easily stored, dosed and standardized. For each of the three prepared batches, no changes in the morphology and integrity of the EVs were observed as a result of the production process; the protein and lipid content as well as the concentration and size distribution of the EVs were reproducible. The chemical-physical analysis, carried out using FTIR, confirmed the simultaneous presence of proteins and lipids in the finished product, while the DSC and TGA analyses showed that the freeze-drying process was successfully completed. Finally, the product met Pharmacopoeia requirements for injectable formulations in terms of sterility, absence of mycoplasma and bacterial endotoxins. In vitro safety and efficacy tests have shown that lyo-secretome stimulates the proliferation of tenocytes, chondrocytes and MSCs. Specifically, the results showed a dose-response effect on cell proliferation (calculated by MTT assay); at the highest concentration of lyo-secretome, corresponding to 400,000 equivalent cells, proliferation reached 85%. Overall, the data obtained represent the premise for the clinical use of lyo-secretome, aimed at establishing its safety and efficacy in equine musculoskeletal diseases (such as tendinitis and osteoarthritis) in animals suffering from spontaneous pathologies.
Le cellule staminali mesenchimali (MSC) sono ormai da diversi anni al centro dell’interesse scientifico mondiale: sia per le incredibili potenzialità che esse presentano dal punto di vista terapeutico, che per le scarse problematiche di natura etica previste per il loro impiego. Negli ultimi decenni è stato dimostrato che l'efficacia terapeutica rigenerativa delle MSC è in gran parte dovuta alla secrezione di un pool di fattori paracrini, denominato secretoma, composto da molecole bioattive libere e solubili tra cui citochine, chemochine e fattori di crescita, e da vescicole extracellulari (EVs) non solubili di dimensioni nano o micrometriche. Il secretoma sembra, quindi, rappresentare una valida alternativa alla terapia cellulare, in quanto offre diversi vantaggi tra cui una minore immunogenicità, maggior applicabilità nella pratica clinica, buona conservabilità e costo accessibile. Anche in ambito veterinario, seppur solo recentemente, l’uso terapeutico del secretoma di MSC in sostituzione delle cellule parentali è stato proposto in clinica, soprattutto per quanto riguarda i disturbi dell’apparato muscolo scheletrico (MSD) che hanno un'alta prevalenza. Tuttavia, è opportuno considerare che l’impiego del secretoma nella pratica clinica, sia umana che veterinaria, è ancora ostacolato dalla mancanza di processi di isolamento scalabili e conformi alle Good Manufacturing Practice (GMP), così come dalla mancanza di formulazione del secretoma in un prodotto medicinale. A tal proposito, questa tesi ha riguardato l’ottimizzazione dello sviluppo tecnologico-farmaceutico di secretoma liofilizzato (lio-secretoma), isolato da MSC equine da tessuto adiposo, la definizione della sua qualità farmaceutica e la valutazione della sua sicurezza ed efficacia in vitro per un potenziale impiego nel trattamento di MSD. Combinando ultrafiltrazione e liofilizzazione, è stato possibile ottenere un prodotto secco a base di secretoma da MSC equine, facilmente conservabile, dosabile e standardizzato. Per la validazione del processo produttivo sono stati preparati tre lotti tecnici in un contesto GMP-compliant. Per ciascuno dei tre lotti, non si sono osservate alterazioni in termini di morfologia e integrità delle EV; il contenuto proteico, lipidico nonché la concentrazione e distribuzione dimensionale delle EV sono risultati riproducibili. L’analisi chimico-fisica, condotta mediante FTIR, ha confermato la simultanea presenza di proteine e lipidi nel prodotto finito, mentre le analisi DSC e TGA hanno dimostrato che il processo di liofilizzazione si è concluso con successo. Infine, il prodotto ha soddisfatto i requisiti di Farmacopea per le formulazioni iniettabili in termini di sterilità, assenza di micoplasma ed endotossine batteriche. I test di sicurezza ed efficacia in vitro hanno dimostrato che il lio-secretoma stimola la proliferazione di tenociti, condrociti e MSC. Nello specifico i risultati hanno mostrato un effetto dose-risposta sulla proliferazione cellulare (calcolata mediante saggio MTT); alla più elevata concentrazione di lio-secretoma, corrispondente a 400.000 cellule equivalenti, la proliferazione raggiungeva l'85%. Nel complesso, i dati ottenuti rappresentano la premessa per l’uso clinico del lio-secretoma, volto a stabilire la sua sicurezza ed efficacia nelle malattie muscoloscheletriche equine (come tendiniti e osteoartriti) su animali affetti da patologie spontanee.
Polvere liofilizzata contenente secretoma di cellule staminali mesenchimali equine: produzione in GMP e attività biologica in vitro.
PONZIO, CHIARA
2019/2020
Abstract
Mesenchymal stem cells (MSCs) have been at the centre of worldwide scientific interest for several years: both because of their incredible therapeutic potential and because of the limited ethical issues involved in their use. In recent decades it has been shown that the regenerative therapeutic efficacy of MSCs is largely due to the secretion of a pool of paracrine factors, called secretome, composed of free and soluble bioactive molecules including cytokines, chemokines and growth factors, and non-soluble extracellular vesicles (EVs) of nano or micrometric size. The secretome seems, therefore, to represent a valid alternative to cell therapy, as it offers several advantages including lower immunogenicity, greater applicability in clinical practice, good shelf life and affordable cost. Even in the veterinary field, although only recently, the therapeutic use of MSC secretome to replace parental cells has been proposed in the clinic, especially for musculoskeletal disorders (MSD) which have a high prevalence. However, it should be considered that the use of secretome in both human and veterinary clinical practice is still hampered by the lack of scalable Good Manufacturing Practice (GMP) compliant isolation processes, as well as the lack of secretome formulation in a medicinal product. In this regard, this thesis concerned the optimization of the technological-pharmaceutical development of lyophilized secretome (lyo-secretome), isolated from equine MSC from adipose tissue, the definition of its pharmaceutical quality and the evaluation of its safety and efficacy in vitro for potential use in the treatment of MSD. By combining ultrafiltration and freeze-drying, it has been possible to obtain a dry product based on equine MSC secretome, easily stored, dosed and standardized. For each of the three prepared batches, no changes in the morphology and integrity of the EVs were observed as a result of the production process; the protein and lipid content as well as the concentration and size distribution of the EVs were reproducible. The chemical-physical analysis, carried out using FTIR, confirmed the simultaneous presence of proteins and lipids in the finished product, while the DSC and TGA analyses showed that the freeze-drying process was successfully completed. Finally, the product met Pharmacopoeia requirements for injectable formulations in terms of sterility, absence of mycoplasma and bacterial endotoxins. In vitro safety and efficacy tests have shown that lyo-secretome stimulates the proliferation of tenocytes, chondrocytes and MSCs. Specifically, the results showed a dose-response effect on cell proliferation (calculated by MTT assay); at the highest concentration of lyo-secretome, corresponding to 400,000 equivalent cells, proliferation reached 85%. Overall, the data obtained represent the premise for the clinical use of lyo-secretome, aimed at establishing its safety and efficacy in equine musculoskeletal diseases (such as tendinitis and osteoarthritis) in animals suffering from spontaneous pathologies.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/11749