Development of 3D in vitro platforms for the study of muscle function and axonal growth and regeneration

Abstract

[eng] The use of in vitro models in biomedical research offers an invaluable tool for exploring biological processes of health and disease. While animal models remain highly informative and necessary to ensure clinical research safety, laboratory models are essential for reducing animal experimentation and enabling fine manipulation and high- throughput analysis. However, there is a concern that traditional in vitro models, based on two-dimensional cultures of mammalian cells and relying on primary culture and non- human cell lines, could lead to inaccurate or misleading results, ultimately jeopardizing the success of novel therapies in clinical trials. Thankfully, exponential advancements in biomedical and biotechnological research have facilitated the creation of increasingly complex and biomimetic models, leading to a paradigm shift in this field. The first pillar of this shift is the use of three-dimensional culture to recapitulate the extracellular matrix environment, through the use of scaffold-based models, lab-on chip devices, and organotypic slice cultures. The second pillar involves transitioning to human cell-derived models to achieve clinically relevant outcomes. The combination of these models with optical techniques such as optogenetics and calcium imaging enables precise manipulation and analysis of cell activity, opening new possibilities for relevant biomedical research. In this work, our general objective was to develop and utilize 3D in vitro models of excitable tissues for two main purposes. On the one hand, our objective was to develop a 3D platform for studying muscle physiology, utilizing human immortalized myoblasts as a clinically relevant cell source. Our next focus was to use this system to model the autoimmune neuromuscular disease myasthenia gravis (MG). On the other hand, we were interested in studying the effect of neuronal activity on axonal regeneration in central nervous system (CNS) neurons. For this, we sought to develop a 3D axotomy platform which could complement previous results obtained in 2D culture. In parallel, we wished to investigate the effect of neuronal activity in a previously established 3D model, namely entorhino-hippocampal organotypic slice cultures (OSCs). In both models, we were interested in applying optogenetic stimulation to Channelrhodopsin-2 (ChR2)-modified neurons.

In the first chapter, we show the validation of in vitro culture systems for human myoblast differentiation. First, we used 2D cultures with hydrogel overlays as an approach for

comparing differentiation and functionality of human myoblast cell lines, as well as for evaluating MG serum antibody binding to in vitro endplates. Later, we developed an anchored 3D in vitro platform for the culture of aligned, differentiated, and contractile human muscle, which responded to electrical, chemical, and optogenetic stimulation. Muscle function was analyzed using motion analysis algorithms or by calcium imaging. Treatment with MG patient serum recapitulated endplate destruction observed in vivo, and some functional effects were observed, although further experiments are needed.

In the second chapter, we developed a 3D axotomy platform using embryonic cortical explants cultured in collagen gels. This model provided a reliable source of axonal projections for axotomy, in contrast to stem-cell derived cultures, which were also evaluated as potential neuronal sources. Using this axotomy platform, we observed that optogenetic stimulation was detrimental to axonal regeneration. In the third chapter, we used entorhino-hippocampal OSCs for a similar purpose, benefitting from our research group's experience in this area. This model recapitulates key neurobiological processes and forms near-in vivo glial scars after lesion; making it a biologically relevant model of CNS development and lesion. We applied optogenetic stimulation on developing connections and axotomized cultures, to study the effect of neuronal activity on axonal pathfinding and regeneration after lesion. We observed that increased neuronal activity resulted in loss of target specificity in developing axons, which could affect regenerative potential in lesioned neurons. In line with the second chapter, optical stimulation was also found to impair axonal regeneration in this model.