An in vitro culture platform for smallcaliber tubular biological structures Bono, Nina (LBB, Laval University, Quebec City, Canada and μBS Lab, Politecnico di Milano, Milan, Italy) Piola, Marco (μBS Lab, Politecnico di Milano, Milan, Italy) Soncini, Monica (μBS Lab, Politecnico di Milano, Milan, Italy) Mantovani, Diego (LBB, Laval University, Quebec City, Canada) Fiore, Gianfranco Beniamino (μBS Lab, Politecnico di Milano, Milan, Italy)

Introduction In the field of tissue engineering, the use of bioreactors plays an important role in the development of mechano-compatible engineered grafts. In this work we present the design and the functional assessment of an in vitro culture platform for small-caliber tubular biological structures, such as native and engineered arterial models (EAMs). The device improves an existing prototype [1], and is designed to allow in vitro culture of tubular structures in a controlled and repro environment (culture mode); in addition, the device integrates a mechanical testing mode for the evaluation of the compliance and burst pressure of the hosted samples (testing mode).

Materials and Methods he culture system (Fig. 1, A) consists of: i) a chamber (a vessel housing inserted into a 50-ml tube acting as reservoir) for the housing of tubular structures of different length and diameters; ii) a hydraulic circuit (silicone tubing) equipped with actuators (a pump and a solenoid pinch-valve); iii) a monitoring and control system (M/C) for the mechanical stimulation and the biomechanical characterization of the hosted samples. The M/C system is designed to ensure the perfusion of the hosted vessels responsible for the wall shear stress, or to impose a controlled physiological-like pulsatile stimulation, resulting in a pressure-related cyclic strain. The M/C system involves a custom LabView software, which manages the hydraulic actuators and operates via a pressure-based feedback control (Fig. 1, B). The M/C also permits evaluating the biomecha properties of hosted samples. During the vessel infusion, the pump processe controlled

flow rate, while measuring the intraluminal pressure. The recorded data are used to estimate the compliance or measure the burst pressure of the vessels. Silicone sleeve supports were used to impart mechanical strain to the hosted vessels (Fig.1, C). Strain-pressure relationship of sleeves (40-mm-length; n=6) were obtained by mounting the sleeves within the culture chamber, and pressurizing them within the range 0-700 mmHg, while digital images of the sleeves were acquired. A semi-empirical model was developed to predict the actual strain of th sam mounted over the sleeves. The data obtained from the characterization tests performed on the sleeves and the actual dimensions and the compliance of the biological samples were used in the model to define a priori the pressure range needed to obtain t desired % strain of the biologic samples (Fig.1, D). To test the reliability of the device in testing mode, functional tests were performed on porcine coronary arteries (n=6). After 3 cycles of preconditioning (0-120 mmHg, step 20 mmHg), the vessels were pressurized at 4 ml/min flow rate until rupture. Preliminary in vitro tests were carried out on EAMs with the device in culture mode Smallcaliber collagen-based EAMs were fabricated in a tubular shape directly within the bioreactor chamber (Fig. 3), following a previously protocol [2]. After fabrication, EAMs were cultured in static conditions up to 7 days.

Results Silicone sleeve characterization tests showed that the sleeves were exposed to approximately 400 mmHg to produce physiological 10% cyclic change in outer diameter (Fig. 1, D). Functional tests demonstrated that cyclic strain may be imposed with accuracy, allowing to modulate properly the stimulation pattern (Fig. 2, D). Preliminary biomechanical characterizations performed on native arteries showed a compliance of (5.0 ± 0.7) x 10^-3 mmHg^-1 and an average burst pressure of 1986 ± 372 mmHg (data comparable with literature [3]) (Fig 2, C). Preliminary in vitro culture tests demonstrate that the specifications of eas fabrication of Ams was satisfied. The system provided a monitored controlled sterile environment sterile environment up to 7 days of culture.

Discussion and Conclusion In this study a flexible and versatile in vitro culture system is presented. Th device is able to integrate a mechanical testing unit for tissue biomec characterization within a culture system where tubular structures are conditioned. The

strategy adopted for the cyclic circumferential strain of the hosted vessels involves the use of a well-characterized silicone support sleeve. This strategy avoids any damage to the vessels cultured over the sleeve, thus extending the potential use of the system to water-permeable tubular structures. In conclusion, the device will be a promising laboratory-oriented tool for stimulating the 3D regeneration of engineered arterial tissues. In this regard, the designed cultur system will be useful to dissect the contribution of different biomechanical factors on tissue architecture and biomechanical characteristics.

Fig. 1: A) Layout of the culture system. B) Pressure stimulation diagram. C) Diagram of the cyclic stimulation strategy. D) Strain vs pressure relationship of silicone sleeves (outer surface) and

nativevessels mounted over the sleeve (outer and inner surfaces): results of the semi-empirical model.

Fig. 2: Photos of the bioreactor during the assembling phase (A) and during the func assessment (B). Example of pressure-volume relationship for one porcine coronary artery mountedwithin the

bioreactor (no sleeve) (C). Pressure and strain tracing at different frequencies (D).

Fig. 3. A. Fabrication of the engineered arterial models within the bioreactor chamber. B.

Staticmaturation of the engineered arterial models. C. Harvesting of the engineered arterial models.

Acknowledgements Bono N. was awarded of a PhD Scholarship from the Italian Ministry of Education and a mobility scholarship from Scuola Interpolitecnica di Dottorato, Italy.

References [1] Piola, M., Prandi, F, et al. A compact and automated ex vivo vessel culture system for t pulsatile pressure conditioning of human saphenous veins. Journal of Tissue Engineering a Regenarative Medicine, 2013, DOI: 10.1002/term.1798. [2] Konig G, McAllister TN, Dusser N, Garrido S, Iyican C, et al. Mechanical properties of completely autologous human tissue engineeed blood vessels compared to human saphenous vein a mammary artery.” Biomaterials, 30(8): 1542-1550, 2009. [3] S. Meghezi, et al. Engineering 3D cellularized collagen gels for vascular tissue regeneration. Journal of Visualized Experiments (In-Press)