Development of tunable bioinks to fabricate 3D-printed in vitro models: a special focus on skeletal muscle models with potential applications in metabolic alteration studies

Abstract

In vitro engineered three-dimensional tissue models are attracting an increasing interest due to their potential applications in preclinical assays. On the one hand, they are an alternative to the high costs, ethical issues and time-consuming experiments associated with animal models. On the other hand, unlike traditional monolayer cultures, 3D models are fabricated with polymer matrices that can mimic the spatial organization and physiological environment of native tissue. The scalability of these models to the market is currently limited by the fabrication methods. Additive manufacturing techniques, as extrusion bioprinting, provide the automated and controlled deposition of biomaterials with encapsulated cells to fabricate 3D models with unlimited shapes. However, few biomaterials fulfill the rheological, mechanical and biological needs for tissue engineering approaches. Printable biomaterials are commonly highly concentrated viscous fluids that could resemble the mechanical properties of stiff tissues, as skeletal muscle. However, they result in restrictive matrices with closed pores that can hamper the migration and proliferation of cells. As a solution, biomaterials have been chemically modified to obtain photocrosslinkable hydrogels, which provide 3D cultures with flexible physical properties. Nevertheless, current bioprinted muscle tissue models show poorly differentiated fibers and lack of functionality. Based on these precedents, this thesis is focused on the development of photocrosslinkable bioinks with tunable physical properties to fabricate customized in vitro 3D models of skeletal muscle tissue and neuroblastoma. To that end, gelatin, alginate and cellulose natural polymers are chemically modified to obtain UV- crosslinkable hydrogels with disparate physical properties. It is found that composite biomaterials of gelatin methacryloyl and alginate methacrylate present the best mechanical properties for stiff tissues as skeletal muscle and tumors. On this basis, the physical properties and composition of GelMA-AlgMA are modified to obtain matrices that resemble the physiological conditions of each tissue. It is found that neuroblastoma models require dense polymer networks to mimic the restrictive matrices found in solid tumors of high-risk patients. Neuroblastoma cell clusters in bioprinted cultures with a high concentration of AlgMA display the characteristic phenotype of aggressive solid tumors. Therefore, this bioink is proposed for the fabrication of stiff neuroblastoma tumor models. In contrast, this formulation was found unsuitable for skeletal muscle models, which present low proliferation and differentiation. Instead, the physical properties of GelMA-AlgMA bioink are tuned by changing the fabrication parameters, and fibrin is added to the composition to obtain a highly porous bioprinted model that resembles the mechanical properties of muscle tissue. As a result, muscle precursor cells are spontaneously differentiated into highly aligned mature fibers that, in combination with an electric pulse stimulation system, develop into mature muscle fibers with contraction capability and pronounced sarcomere units. The functionality of the bioprinted tissue agrees with the metabolic activity analysis, which corresponds to the behavior of native tissue. Hence, this model could be used to monitor the effects of drugs in the metabolic respiration of muscle mitochondria, simplifying the traditional protocols based on the isolation of single fibers. As an approach to obtain faithful platforms for the study of muscle pathologies as cancer cachexia, bioprinted muscle rings are treated with medium conditioned with colorectal

cancer cells. This study shows that soluble factors secreted by cancer cells induce the upregulation of protein degradation pathways and degeneration of muscle fibers. In particular, high levels of soluble TNFRI are associated with severe cachexia, which correlates with the plasma level of tumor-bearing mice. The results indicate a close resemblance between the gene expression pattern of the bioprinted model and muscle tissue of cachectic mice, whereas monolayer cultures present several disparities. Together, GelMA-AlgMA-Fibrin is presented as a promising biomaterial for the fabrication of bioprinted models of healthy skeletal muscle tissue and muscle wasting on cancer cachexia disease.

La bioimpresión por extrusión es una técnica prometedora para el escalado y la fabricación automatizada de modelos tisulares in vitro. Sin embargo, las propiedades fisicoquímicas de las biotintas actuales pocas veces cumplen los requisitos de los tejidos naturales. Esta tesis describe la modificación química de polímeros naturales como gelatina y alginato para obtener biotintas fotopolimerizables con propiedades físicas maleables. La composición de las biotintas y los parámetros de fabricación de los diseños bioimpresos son modulados para conseguir dos tipos de formulaciones adecuadas para el desarrollo de modelos de músculo esquelético y neuroblastoma. En el estudio, ambos modelos muestran mayor semblanza a los tejidos originales que los tradicionales cultivos en monocapa. Por un lado, los modelos de neuroblastoma recapitulan el comportamiento de los tumores sólidos en pacientes de alto riesgo. Por otro lado, la composición de la biotinta para modelos musculares induce la diferenciación de células precursoras a fibras musculares maduras y alineadas que, en combinación con un sistema de estimulación por pulsos eléctricos, dan como resultado fibras musculares funcionales con capacidad contráctil. Este modelo imita el comportamiento del músculo esquelético y se puede utilizar para monitorizar los efectos de compuestos químicos sobre el tejido. Al final del estudio, se trabaja en la generación de un modelo de músculo atrofiado por caquexia derivada del cáncer. El modelo bioimpreso reproduce la característica sobreexpresión de genes relacionados con la degradación proteica hallada en ratones. En conjunto, se adaptan la composición y propiedades físicas de biotintas basadas en gelatina y alginato para la fabricación de modelos de tejido muscular y neuroblastoma por medio de fabricación aditiva. Además, el modelo muscular se presenta como un posible candidato para realizar estudios in vitro del tejido sano y atrofiado por caquexia derivada de cáncer.