Characterization and Optimization of Cell Seeding in Electrospun Scaffolds by Mean of a Micro-Optofluidic Bioreactor

Nowadays the demand for tissue regeneration is growing due to a scarcity of donors and biocompatibility issue in transplant immune rejection. For these reasons, scientists are investigating artificial tissues as alternative to regenerate damaged ones by combining cells with nanostructured scaffolds (Park et al. 2016). Scaffolds play a crucial role in tissue engineering, working as temporary skeleton for cells adhesion and proliferation when a tissue is damaged. Thus, in this context, the first step of cell culture within scaffolds is the cell seeding (Luyten et al. 2001). From the latter depends the initial cells quantity and their spatial arrangement, affecting in turn cell proliferation, migration, and the differentiation of the engineered implant (Schliephake et al. 2009). Hence, the cell seeding represents a crucial step in the development of efficient construct for regeneration purposes. In this context arise the need for the development of reliable seeding techniques. Among these, according to the literature, have been used static, rotational and perfusion protocols. The former is the most used one, consisting of passively introducing concentrated cell suspension into a scaffold placed in a well. However, some drawbacks are associated to this approach. First, depending on the scaffold’s density, these can float or move within wells during culture or media changes, making it difficult to maintain a consistent microenvironment. Second, if the used material is hydrophobic, providing the cells by using a pipette, the cell adhesion can be hindered. Third, the 3D porous constructs can interfere with conventional imaging techniques, so complicating quantitative analysis (cell counting). In this context, this study aims to introduce a micro-optofluidic (MoF) bioreactor, characterized by an encapsulated polyether sulfone scaffold, for cell seeding performed in a dynamic way to achieve good cell seeding efficiency (CSE) and cell spatial distribution (CSD). Thus, the latter responses were investigated by mean of a Design of Experiments (DoE) approach, where as influential factors were selected the microparticles type, the f low rate, imposed to feed the scaffold with the concentrated cell suspension, and the seeding time. The selected factors belong them affecting the quality of the seeded scaffold (Impens et al. 2010). In detail, the scaffold was fabricated via electrospinning, because it is a simple and cost-effective technique to realize non-woven polymeric architecture of nanofibers morphologically similar to the native extra-cellular matrix (Keshvardoostchokami et al. 2020), with a high surface-to-volume ratio and microporous morphology enabling cells adhesion. Next, the MoF device was realized using a 3D printing-based master/slave approach (Cutuli et al. 2023). It was made of polydimethylsiloxane for two reasons: it is characterized by nontoxicity and good gas permeability, which makes it suitable for biomedical applications as bioreactor in cell culture; it is transparent, so enabling its use for the optical detection. This feature is crucial since the two investigated responses, i.e., CSE and CSD were monitored, while varying in a systematic way all the selected design factors, by using an optical approach relying on the optical absorption (using optical fibers) and imaging techniques. The former relied on the investigation of acquired optical signals, whilst the latter on an image analysis approach.