Screening of perfused combinatorial 3D microenvironments for cell culture.

Biomaterials combining biochemical and biophysical cues to establish close-to-extracellular matrix (ECM) models have been explored for cell expansion and differentiation purposes. Multivariate arrays are used as material-saving and rapid-to-analyze platforms, which enable selecting hit-spotted formulations targeting specific cellular responses. However, these systems often lack the ability to emulate dynamic mechanical aspects that occur in specific biological milieus and affect physiological phenomena including stem cells differentiation, tumor progression, or matrix modulation. We report a tailor-made strategy to address the combined effect of flow and biochemical composition of three-dimensional (3D) biomaterials on cellular response. We suggest a simple-to-implement device comprising (i) a perforated platform accommodating miniaturized 3D biomaterials and (ii) a bioreactor that enables the incorporation of the biomaterial-containing array into a disposable perfusion chamber. The system was upscaled to parallelizable setups, increasing the number of analyzed platforms per independent experiment. As a proof-of-concept, porous chitosan scaffolds with 1 mm diameter were functionalized with combinations of 5 ECM and cell-cell contact-mediating proteins, relevant for bone and dental regeneration, corresponding to 32 protein combinatorial formulations. Mesenchymal stem cells adhesion and production of an early osteogenic marker were assessed on-chip on static and under-flow dynamic perfusion conditions. Different hit-spotted biomaterial formulations were detected for the different flow regimes using direct image analysis. Cell-binding proteins still poorly explored as biomaterials components - amelogenin and E-cadherin - were here shown as relevant cell response modulators. Their combination with ECM cell-binding proteins - fibronectin, vitronectin, and type 1 collagen - rendered specific biomaterial combinations with high cell adhesion and ALP production under flow. The developed versatile system may be targeted at widespread tissue regeneration applications, and as a disease model/drug screening platform. STATEMENT OF SIGNIFICANCE: A perfusion system that enables cell culture in arrays of three-dimensional biomaterials under dynamic flow is reported. The effect of 31 cell-binding protein combinations in the adhesion and alkaline phosphatase (ALP) production of mesenchymal stem cells was assessed using a single bioreactor chamber. Flow perfusion was not only assessed as a classical enhancer/accelerator of cell growth and early osteogenic differentiation. We hypothesized that flow may affect cell-protein interactions, and that key components driving cell response may differ under static or dynamic regimes. Indeed, hit-spotted formulations that elicited highest cell attachment and ALP production on static cell culture differed from the ones detected for dynamic flow assays. The impacting role of poorly studied proteins as E-cadherin and amelogenin as biomaterial components was highlighted.

Tuneable spheroidal hydrogel particles for cell and drug encapsulation.

The need to better mimic native tissues has accompanied research in tissue engineering and controlled drug delivery. The development of new platforms for cell and drug encapsulation followed the same trend, and studying the influence of the delivery material system's geometry has been gaining momentum. Aiming to investigate how an increase in surface area and varying particle shape could impact drug release and cell viability, a novel method was developed to produce spheroidal hydrogel particles with adjustable circularity, aiming to tune drug delivery. For this purpose, droplets of hydrogel precursor were squeezed between two superamphiphobic surfaces separated with spacers with different height, and then photo-crosslinked to maintain the acquired shape after "de-sandwiching". Numerical modelling studies were performed to study the polymeric droplet geometry deformation process, which were consistent with experimentally obtained results. The spheroidal particles were produced under mild conditions using methacrylated chitosan, capable of encapsulating proteins or cells. Likely due to their higher surface area to volume-ratio, compared to spherical-shaped ones, spheroids presented an improved viability of encapsulated cells due to enhanced nutrient diffusion to the core, and led to a significantly faster drug release rate from the polymer network. These results were also assessed numerically, in which the drug release rate was computed for different spheroidal-like geometries. Hence, the described method can be used to manufacture spheroidal particles with tailored geometry that can be broadly applied in the biomedical field, including for drug delivery or as cell encapsulation platforms.

Bioinspired methodology for preparing magnetic responsive chitosan beads to be integrated in a tubular bioreactor for biomedical applications.

Magnetic responsive chitosan beads were prepared using a methodology inspired by the rolling of water droplets over lotus leaves. Liquid precursors containing chitosan and magnetic microparticles were dispensed in the form of spherical droplets and crosslinked with genipin over synthetic superhydrophobic surfaces. Scanning electronic microscopy, histology and micro-computed tomography were employed to characterize the structure of the prepared composite beads and the inner distribution of the magnetic particles. Cellular metabolic activity tests showed that fibroblasts-like (L929 cell line) can adhere and proliferate on the prepared chitosan beads. We hypothesize that such spherical biomaterials could be integrated in a new concept of tubular bioreactor. The magnetic beads can be immobilized by an external magnetic field at specific positions and may be transported along the bioreactor by the drag of the culture medium flow. The system behavior was also studied through numerical modeling, which allowed to identify the relative importance of the main parameters, and to conclude that the distance between carrier beads plays a major role on their interaction with the culture medium and, consequently, on the overall system performance. In an up-scaled version of this bioreactor, the herein presented system may comprise different chambers in serial or parallel configurations. This constitutes a simple way of preparing magnetic responsive beads combined with a new design of bioreactor, which may find application in biomedicine and biotechnology, including in cell expansion for tissue engineering or for the production of therapeutic proteins to be used in cell therapies.