Design and validation of a 3D-bioprinted, dynamically cultured in vitro model of the liver

INTRODUCTION Continuous advances in the field of biofabrication are providing new tools for the production of progressively more complex in vitro models, thus reinforcing the belief that this kind of platforms could play a major role in increasing not only the throughput of research in the life sciences, but also the degree of personalization of modern medicine [1], [2]. In this context, in vitro models of the liver are showing their potential both when exploited for fundamental biological research, and when coupled with animal experimentation during drug development. With this in mind, this works describes the development of a 3D-bioprinted in vitro model of the hepatic environment, specifically conceived to recapitulate the set of chemomechanical stimuli that hepatic cells are subjected to, aiming to enhance the adherence of their behavior and functionality to the ones observed in vivo. MATERIALS AND METHODS The bioink to produce the model is based on an alginate-hepatic extracellular matrix (ECM) powder hydrogel, aiming to provide cultured cells with an in vivo-like set of chemomechanical stimuli. The viscoelastic properties of the material were tuned to match the ones of a physiological porcine models, that were measured through tailored rheological tests (MCR502, Anton-Paar, AT). ECM was extracted from porcine livers through a tailored decellularization protocol, freeze-dried, powdered, and tested in terms of residual biological macromolecules. The printability of the material was studied a priori through different specific rheological tests, and empirically, by printing it with a pneumatic bioprinter (BioX, CELLINK, SE). The tuning of printing parameters to manufacture 3D constructs was performed by analyzing and optimizing major shape-fidelity coefficients (fiber uniformity, pore coefficient, perimeter coefficient, and printability coefficient) of fibers and grid structures printed in different conditions and with different needles. Finally, a 10x10x3 mm3 parallelepipedal geometry, was printed with a 15% grid infill to be dynamically cultured. HepG2 cells were embedded within the bioink at a final concentration of 3x106 cells/ml. Cellularized constructs were statically cultured up to 8 days, aiming to study their ability to sustain 3D cell cultures. Two other bioinks, one made with only alginate, and the other with a blend of alginate and gelatin were used as controls, to assess potential beneficial effects induced by the presence of ECM. RESULTS AND DISCUSSION After decellularization, type I collagen, elastin, fibronectin, and glycosaminoglycans were preserved, without significant differences from a native sample. The preservation of these macromolecules is crucial since they can deeply influence cell behavior in terms of functionality, adhesion, detachment, and proliferation [3], [4]. This, coupled with the mimicry of the viscoelastic properties of the physiological tissue ensures a chemomecanically favorable environment to 3D culture hepatic cells. A priori rheological analyses highlighted the presence of yielding point (from which the loss modulus G’’ of the material started prevailing over the elastic modulus G’) at a shear stress value of ≈ 60 Pa, thus indicating the possibility to extrude the bioink. Moreover, a recovery test showed that the material is able to recover its original viscoelastic properties after the extrusion process [5]. Trial and error procedure revealed that the optimal setting to print the designed geometry included a 27 G conical nozzle, implementing a 23 kPa extrusion pressure, and a 6 mm/s printhead speed. The printed geometry was found to be stable for the whole experimental window in the dynamic culture conditions. Preliminary results from 8 days static cultures revealed that the presence of ECM positively impacted cell viability by increasing it up to 400%, if compared to controls. Ongoing studies include the development of a computational model to optimize the set-up of a bioreactor, in which dynamically culture 3D-bioprinted constructs, while monitoring their viability and metabolic activities. CONCLUSIONS The presented bioink mimics the physiological chemomechanical features of the hepatic environment. Preliminary viability results represent a proof of concept of the ability of the developed material to sustain cell proliferation in the mid-term.