Engineered functional 3D spheroids for β-cell encapsulation

Abstract

[eng] Type 1 Diabetes Mellitus (T1DM) is known to be associated with the immune-mediated destruction of insulin-secreting β-cells. Cell replacement therapy is thought to be a potential long-lasting cure for diabetes. In this regard, allogeneic pancreatic islet transplantation is currently considered an alternative therapeutic option. However, the shortage of islet donors and the poor islet survival after transplantation restrict the use of this technique. Human embryonic (hESC) stem cell differentiation techniques have enabled the large-scale in vitro production of stem cell-derived β cells (SC-βs), for future cell replacement therapy. Even so, graft rejection can occur as a result of the innate and adaptive immune response, necessitating chronic immunosuppressive regimes. A key component in successfully optimizing graft survival after transplantation is immune regulation, and the rapid establishment of blood flow for nutritional and oxygen supply. In this line, the application of immunosuppressive medication can be avoided when transplanted cells are embedded in immunoprotective membranes. These membranes can protect cells from the host immune system and provide a 3D architecture emulating the microenvironment found in vivo. The encapsulation in immunoprotective membranes can be done in two geometries, i.e., macro- and microdevices. The advantage of microdevices, such as microspheres lies in their high surface-to-volume ratio, which facilitates faster oxygen diffusion and the exchange of glucose, insulin, and nutrients. Further, microspheroids or spheroids can be easily handled in a fluid suspension, and precise spatial and dosing controls are possible for in vivo applications. Current microsphere fabrication technologies have several drawbacks such as the need for an oil phase containing surfactants, diameter heterogeneity of the microspheres, and high time-consuming processes. Moreover, these methodologies frequently use alginate, which has low biocompatibilities and can lead to a fibrotic response, thereby limiting the efficacy of the graft. The use of collagen-based hydrogels is becoming increasingly recognized as an ideal biomaterial for cell-laden hydrogels. It is a natural material with good biological compatibility and low antigenicity. However, collagen is biodegradable, is challenging to handle and the fibrillar structure is weak for direct use. Consequently, we propose in this thesis a strategy for optimizing the cell delivery device. The first aim of this thesis was to develop a novel methodology to produce microspheres in an automated fashion, able to mask the encapsulated cells from the immune system, without adversely affecting cell viability. We generated three-dimensional (3D) bioprinted collagen microspheres by deposition of the cell-laden bioink into a superhydrophobic surface. Recent studies have proposed collagen crosslinkers that can confer mechanical firmness to collagen hydrogel. However, their plethora of disadvantages ranges from cytotoxicity to expensive cost and excessive crosslinking time. Here, we have crosslinked the bioprinted microspheres with tannic acid (TA) which improved spherical and structural consistency. Our results have also uncovered that TA prevents collagenase degradation and enhances spherical structural consistency and stiffness while allowing the diffusion of insulin. The second aim of this thesis was to develop a pancreatic tissue-derived decellularized matrix hydrogel to obtain the extracellular matrix (ECM) properties to faithfully recreate the microenvironment of the islets in vivo. The use of decellularized ECM (dECM) scaffolds for bioengineering of human-tissue constructs is envisioned as a major platform for therapeutic applications. Here, we demonstrated an in-house optimal human decellularization protocol to generate a human dECM hydrogel. The developed hydrogel represented the possibility of engineering functional human insulin-secreting tissue constructs.

[cat] La diabetis mellitus tipus 1 (T1DM) és una malaltia que es deu a un dèficit d'insulina. Aquest dèficit neix com a resultat de la destrucció de les cèl·lules β del pàncrees per part del sistema immunitari. Les tècniques de diferenciació de cèl·lules mare embrionàries humanes (hESC), han permès la producció in vitro a gran escala de cèl·lules β, productores d’insulina. La medicació immunosupressora crònica es podria evitar encapsulant les cèl·lules en microesferes composades per membranes immunoprotectores; aquestes membranes poden protegir les cèl·lules del sistema immunitari del hoste, i proporcionar una arquitectura 3D que emuli el microambient trobat in vivo. A més a més, les microesferes suspeses en fluid permeten ser injectades fàcilment en el cos del hoste. El primer objectiu d'aquesta tesi va ser desenvolupar una nova metodologia per produir microesferes de manera automatitzada. Una tecnologia capaç d'emmascarar les cèl·lules encapsulades del sistema immunitari, sense afectar negativament la viabilitat cel·lular. Aquesta nova tecnologia consisteix en la generació de microesferes mitjançant l’ús d’una bioimpresora 3D. L’hidrogel en solució, que conté les cèl·lules β, es diposita en volums controlats en una superfície hidròfoba. Això fa que l’esfericitat de la microesfera es mantingui i genera la possibilitat de controlar el volum; proporcionant microesferes de diàmetres homogenis. Seguidament per conferir més fermesa a l’estructura, les microesferes es van crosslinkar amb acid tànic (TA). Els nostres resultats demostren que el TA evita que la col·lagenasa degradi el col·lagen i permet la difusió de la insulina per part de les cèl·lules encapsulades. El segon objectiu d'aquesta tesi va ser generar un hidrogel que recreés fidelment, no només el microambient on es troben els illots pancreàtics in vivo, sinó també els senyals moleculars que els influeixen. La descel·lularització; una nova tecnologia que permet extreure les cèl·lules d’un òrgan, per poder aïllar la matriu extracel·lular (ECM). Mitjançant aquesta tecnologia vam poder generar un hidrogel a partir d’un pàncreas humà de donant. L’hidrogel amb les propietats de la ECM del pàncrees ens va permetre encapsular cèl·lules β humanes, generat així un teixit funcional capaç de segregar insulina.