High-Fidelity Extrusion Bioprinting of Low-Printability Polymers Using Carbopol as a Rheology Modifier.

Extrusion three-dimensional (3D) bioprinting is a promising technology with many applications in the biomedical and tissue engineering fields. One of the key limitations for the widespread use of this technology is the narrow window of printability that results from the need to have bioinks with rheological properties that allow the extrusion of continuous filaments while maintaining high cell viability within the materials during and after printing. In this work, we use Carbopol (CBP) as rheology modifier for extrusion printing of biomaterials that are typically nonextrudable or present low printability. We show that low concentrations of CBP can introduce the desired rheological properties for a wide range of formulations, allowing the use of polymers with different cross-linking mechanisms and the introduction of additives and cells. To explore the opportunities and limitations of CBP as a rheology modifier, we used ink formulations based on poly(ethylene glycol)diacrylate with extrusion 3D printing to produce soft, yet stable, hydrogels with tunable mechanical properties. Cell-laden constructs made with such inks presented high viability for cells seeded on top of cross-linked materials and cells incorporated within the bioink during printing, showing that the materials are noncytotoxic and the printed structures do not degrade for up to 14 days. To our knowledge, this is the first report of the use of CBP-containing bioinks to 3D-print complex cell-laden structures that are stable for days and present high cell viability. The use of CBP to obtain highly printable inks can accelerate the evolution of extrusion 3D bioprinting by guaranteeing the required rheological properties and expanding the number of materials that can be successfully printed. This will allow researchers to develop and optimize new bioinks focusing on the biochemical, cellular, and mechanical requirements of the targeted applications rather than the rheology needed to achieve good printability.

3D Bioprinting Strategies, Challenges, and Opportunities to Model the Lung Tissue Microenvironment and Its Function.

Human lungs are organs with an intricate hierarchical structure and complex composition; lungs also present heterogeneous mechanical properties that impose dynamic stress on different tissue components during the process of breathing. These physiological characteristics combined create a system that is challenging to model in vitro. Many efforts have been dedicated to develop reliable models that afford a better understanding of the structure of the lung and to study cell dynamics, disease evolution, and drug pharmacodynamics and pharmacokinetics in the lung. This review presents methodologies used to develop lung tissue models, highlighting their advantages and current limitations, focusing on 3D bioprinting as a promising set of technologies that can address current challenges. 3D bioprinting can be used to create 3D structures that are key to bridging the gap between current cell culture methods and living tissues. Thus, 3D bioprinting can produce lung tissue biomimetics that can be used to develop in vitro models and could eventually produce functional tissue for transplantation. Yet, printing functional synthetic tissues that recreate lung structure and function is still beyond the current capabilities of 3D bioprinting technology. Here, the current state of 3D bioprinting is described with a focus on key strategies that can be used to exploit the potential that this technology has to offer. Despite today's limitations, results show that 3D bioprinting has unexplored potential that may be accessible by optimizing bioink composition and looking at the printing process through a holistic and creative lens.

A Robust Protocol for Decellularized Human Lung Bioink Generation Amenable to 2D and 3D Lung Cell Culture.

Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems.