Tissue Engineering (TE) is a branch of regenerative medicine and a broad definition for any therapy that aims to develop cell-based tissue substitutes in order to restore, reconstruct, or improve tissue functions. TE employs different approaches as direct cell implantation, in situ tissue regeneration or transplantation of tissues. The procedure for the realization of an engineered tissue requires in first, a donor biopsy, from which cells are isolated and expanded in vitro, followed by seeding on appropriate 3D supports. Good biological substrates currently available are decellularized ExtraCellular Matrices (ECM). Decellularization is a technique used to create a scaffold removing the cellular material from a tissue, maintaining the ECM. This is a very promising field, because it can be applied to any type of tissue, of animal or vegetal origin. The use of decellularized matrices has considerable advantages: preservation of the ECM, positive immunogenicity of the scaffold and the creation of a tissue-specific scaffold. Various decellularization protocols have been evaluated; in general they are based on three phases: lithic process (with chemical agents, enzymatic treatments or physical agents), removal of cellular and nucleic components from the ECM and washing of the structure. The efficiency is strictly dependent upon the tissue of interest. The decellularization process has not been defined by quantitative metrics, but the minimal criteria to satisfy the intent of decellularization are: less than 50 ng dsDNA per mg ECM dry weight, less than 200 bp DNA fragment length, lack of visible nuclear material in tissue sections stained with DAPI or H\&E. Two kind of signals can be applied: chemical and physical. The bioreactor is used to generate stimulation that affects the growing cells to enhance specific development of the tissue. This can be used for growing of cells for different applications. Bioreactors are devices in which biological/biochemical processes develop under a closely monitored and tightly controlled environment. This project aims to develop and validate a bioreactor for the perfusion of an immunocompetent bioengineered 3D liver tissue, with metabolic and immunological functionality for disease modeling. A disease model is an animal, cells or \emph{in vitro} model displaying all or some of the pathological processes that are observed in human disease. The bioengineered liver, improved in this project, combined a decellularized whole rat liver with human hepatic cells and matched immune cells (Peripheral Blood Mononuclear cell; PBMCs) and a custom bioreactor for the support of cell survival. A new bioreactor for use in scientific research has been designed and developed. The device was able to maintain sterility after 4 days, as demonstrated by the mycoplasma test. It permitted the live-imaging analysis with the IVIS. Thanks to the right geometric choices and of the materials, the device proved to be suitable for use. The structural and biological characterizations performed on the bioreactor have underlined the ease of use. A decellularization protocol was improved with the air removal system, the scaffold obtained with this device presented a more clear and preserved vasculature. Both the cell seeding with HepG2 and the perfusion with PBMCs showed good results, but they also highlighted limits of the system. Seeding with HepG2 has shown excellent results, in fact in this project was demonstrated the proliferation of the cells with this system. But during the perfusion all three cases showed a reduction in the number of cells. For PBMCs, they alived after two days, but they showed reduced vitality.
L'ingengneria tissutale è una branca della medicina rigenerativa. Essa mira a ripristinare, ricostruire o migliorare le funzioni tissutali, grazie allo sviluppo di sostituti tissutali a base cellulare. L'ingegneria tissutale utilizza il trapianto di cellule, la rigenerazione tissutale in situ o l'impianto di tessuti. Gli scaffold decellularizzati sono considerati tra i più promettenti. La decellularizzazione è una tecnica utilizzata per creare scaffold privi di materiale cellulare, mantenendo la matrice extracellulare (ECM). L'uso di matrici decellularizzate presenta notevoli vantaggi: conservazione dell'ECM, una risposta immunitaria positiva in vivo nell'ospite e creazione di uno scaffold specifico per l'ospite. Sono stati valutati vari protocolli di decellularizzazione; in generale si basano su tre fasi: lisi cellulare (con agenti chimici, o fisici o trattamenti enzimatici), rimozione di componenti cellulari e nucleici dall'ECM e lavaggio. L'efficienza dipende strettamente dal tessuto di interesse. Il processo di decellularizzazione è considerato ben riuscito se sono rispettate le seguenti prerogative: meno di 50 ng dsDNA per mg di ECM a secco, meno di 200 bp di lunghezza dei frammenti di DNA, mancanza di materiale nucleare visibile nelle sezioni di tessuto colorato con DAPI o H\&E. Possono essere applicati due tipi di segnali al costrutto: chimico e fisico. Il bioreattore viene utilizzato per generare una stimolazione che colpisca le cellule in crescita per migliorare lo sviluppo specifico del tessuto. I bioreattori sono dispositivi in cui i processi biologici/biochimici si sviluppano in un ambiente strettamente monitorato e controllato. Questo progetto mira a validare e sviluppare un bioreattore per la perfusione di un fegato bioingegnerizzato e immunocompetente, con funzionalità metaboliche e immunologiche per essere utilizzato come un modello di malattia. Un modello di malattia è modello animale, cellulare o in vitro che mostri tutti o alcuni dei processi patologici osservati in una malattia umana. Questo modello, combina un fegato di ratto decellularizzato con cellule epatiche umane e cellule immunitarie (Cellule Periferiche Mononucleari del Sangue, PBMCs). Il bioreattore viene adoperato per il supporto cellulare. In questo lavoro di tesi è stato progettato e sviluppato un nuovo bioreattore da utilizzare nella ricerca scientifica. Il dispositivo è stato in grado di mantenere la sterilità dopo 4 giorni, come dimostrato dal test al micoplasma. Tale camera ha permesso le analisi all'IVIS, grazie alle giuste scelte geometriche e ai materiali. Il dispositivo si \'e dimostrato idoneo all'uso. Le caratterizzazioni strutturali e biologiche eseguite sul bioreattore hanno sottolineato la facilità d'uso. Il protocollo di decellularizzazione è stato migliorato con il sistema di rimozione dell'aria, lo scaffold ottenuto con questo dispositivo ha presentato una vascolarizzazione più chiara e conservata. Sia la semina cellulare con HepG2 che la perfusione con PBMCs hanno mostrato buoni risultati, ma hanno anche evidenziato i limiti del sistema. La semina con HepG2 ha mostrato risultati eccellenti, infatti la semina è risultata molto promettente con un'adesione cellulare molto alta. Inoltre il picco di radianza media riscontrato al giorno 2 nell'esperimento D003, ha permesso di concludere che il sistema ideato permette la crescita cellulare. Nonostante ciò, la perfusione tutti in tutti e tre i casi ha mostrato una riduzione del numero di cellule. Per le PBMCs, gli esperimenti hanno mostrato la soppravivenza delle cellule ma anche una vitalità ridotta.
Bioreactor based immunocompetent bioengineered 3D liver for disease modeling
SIENA, RICCARDO RAYAN
2017/2018
Abstract
Tissue Engineering (TE) is a branch of regenerative medicine and a broad definition for any therapy that aims to develop cell-based tissue substitutes in order to restore, reconstruct, or improve tissue functions. TE employs different approaches as direct cell implantation, in situ tissue regeneration or transplantation of tissues. The procedure for the realization of an engineered tissue requires in first, a donor biopsy, from which cells are isolated and expanded in vitro, followed by seeding on appropriate 3D supports. Good biological substrates currently available are decellularized ExtraCellular Matrices (ECM). Decellularization is a technique used to create a scaffold removing the cellular material from a tissue, maintaining the ECM. This is a very promising field, because it can be applied to any type of tissue, of animal or vegetal origin. The use of decellularized matrices has considerable advantages: preservation of the ECM, positive immunogenicity of the scaffold and the creation of a tissue-specific scaffold. Various decellularization protocols have been evaluated; in general they are based on three phases: lithic process (with chemical agents, enzymatic treatments or physical agents), removal of cellular and nucleic components from the ECM and washing of the structure. The efficiency is strictly dependent upon the tissue of interest. The decellularization process has not been defined by quantitative metrics, but the minimal criteria to satisfy the intent of decellularization are: less than 50 ng dsDNA per mg ECM dry weight, less than 200 bp DNA fragment length, lack of visible nuclear material in tissue sections stained with DAPI or H\&E. Two kind of signals can be applied: chemical and physical. The bioreactor is used to generate stimulation that affects the growing cells to enhance specific development of the tissue. This can be used for growing of cells for different applications. Bioreactors are devices in which biological/biochemical processes develop under a closely monitored and tightly controlled environment. This project aims to develop and validate a bioreactor for the perfusion of an immunocompetent bioengineered 3D liver tissue, with metabolic and immunological functionality for disease modeling. A disease model is an animal, cells or \emph{in vitro} model displaying all or some of the pathological processes that are observed in human disease. The bioengineered liver, improved in this project, combined a decellularized whole rat liver with human hepatic cells and matched immune cells (Peripheral Blood Mononuclear cell; PBMCs) and a custom bioreactor for the support of cell survival. A new bioreactor for use in scientific research has been designed and developed. The device was able to maintain sterility after 4 days, as demonstrated by the mycoplasma test. It permitted the live-imaging analysis with the IVIS. Thanks to the right geometric choices and of the materials, the device proved to be suitable for use. The structural and biological characterizations performed on the bioreactor have underlined the ease of use. A decellularization protocol was improved with the air removal system, the scaffold obtained with this device presented a more clear and preserved vasculature. Both the cell seeding with HepG2 and the perfusion with PBMCs showed good results, but they also highlighted limits of the system. Seeding with HepG2 has shown excellent results, in fact in this project was demonstrated the proliferation of the cells with this system. But during the perfusion all three cases showed a reduction in the number of cells. For PBMCs, they alived after two days, but they showed reduced vitality.| File | Dimensione | Formato | |
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Riccardo, Rayan, Siena_Thesis.pdf
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Descrizione: M.Sc. Thesis in Biomedical Engineering; Riccardo Rayan Siena
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https://hdl.handle.net/10589/141702