Poster M13 - Perfusion Based System Incorporating Bioprinted Vascularized Tissue for Drug Delivery Applications

Introduction: Recent studies have demonstrated significant advantages that 3D cell culture models provide over their 2D counterparts, including more accurate representations of cell morphology, proliferation gradients, drug response, gene expression, and overall cell behavior [1]. 3D bioprinting has emerged as a viable solution, offering increased efficiency and reproducibility. Another key benefit of this technology is that 3D-printed human tissues can be printed in microfluidic-based chips, serving as an alternative for animal trials [2]. Integration of perfusion systems in 3D bioprinted tissues can enhance nutrient and oxygen supply to cells, replicating physiological conditions more closely. This improvement is critical for developing functional tissues for studying disease mechanisms and testing new treatments under more realistic conditions. Perfusion systems also allow for the continuous removal of metabolic waste, which is essential for maintaining long-term cell viability and function. These microfluidic 3D culture devices, known as Organs-on-chips (OoC), can promote structural complexity and incorporate dynamic fluid flow and mechanical stimulations. The use of animal models has resulted in more than 90% of candidate drugs failing in human trials, primarily due to differences between animal and human physiology [3]. The application of 3D bioprinting and OoC can provide many benefits, including the customization and personalization of medical products, pharmaceutical research regarding forms of drug development, dosage, delivery, discovery, and toxicology testing.

Materials and

Methods: The printing of perfusable channels will be carried out by printing the sacrificial ink (Pluronic F127) onto the bed of the VasKit device. The printing will be performed under pressures in the range of 40-90 kPa to achieve vessels with a 400 µm diameter. To wash out the sacrificial ink and form perfusable channels, the temperature must be lowered to 4°. This will cause the ink to become a liquid that flows readily. The ink can then be removed from the 3D bioprinted construct by flushing the channel. This is done by inserting a syringe into the inlet of the construct and flushing the channel with sterile deionized water. Upon extraction of Pluronic F127, the vessels can be flushed with fluorescent red dye to visualize and confirm proper channel formation. Before seeding the channels with endothelial cells like HUVECs, the inner surfaces are coated with a fibrinogen solution to promote cell attachment and lining of the channels. The peristaltic pump drives continuous media perfusion once the channels are cellularized. This physiologically relevant interstitial flow supports the long-term culture, maturation, and functioning of the bioprinted vascular bed. Overall, this integrated bioprinting and perfusion approach, as presented in Figure 1, enables unprecedented structural and microfluidic control over engineered vascularized tissue models. The viability of the cells within the channels was assessed using the LIVE/DEAD® Viability assay kit.

Results, Conclusions, and Discussions: 3D bioprinted tissue constructs containing perfusable vascular networks were successfully created, as evident in Figure 2. The interstitial flow in this perfusion device facilitated the adherence, proliferation, and confluence of human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted channels over 30 days. Fluorescent staining and confocal microscopy confirmed the formation of intact endothelial monolayers lining the perfusable lumens, with HUVECs exhibiting characteristic rounded, grouped clump morphologies.
This versatile bioprinting and perfusion platform enables the engineering of biomimetic pre-vascularized tissue models with precisely defined 3D microvasculature geometries. These vascularized tissue-engineered constructs have numerous promising applications, including physiologically relevant models for drug delivery and testing. Notably, the potential of microfluidic-based perfusion systems to advance drug development lies in their ability to inform decisions on which drugs should proceed to further development and testing and which should be discontinued by providing humanized drug toxicity information. Furthermore, this approach allows effective drug repurposing for specific patient populations.

References:

1. Ravi, M., Paramesh, V., Kaviya, S. R., Anuradha, E., & Solomon, F. P. (2015). 3D cell culture systems: advantages and applications. Journal of cellular physiology, 230(1), 16-26.
2. Wang, X., Sun, Q., & Pei, J. (2018). Microfluidic-based 3D engineered microvascular networks and their applications in vascularized microtumor models. Micromachines, 9(10), 493.
3. Gintant, G., Sager, P.T., and Stockbridge, N. (2016). Evolution of strategies to improve preclinical cardiac safety testing. Nat. Rev. Drug Discov. 15, 457–471.

Acknowledgements (Optional): The authors would like to acknowledge funding support from National Science Foundation – Grant Number CBET 2317757.