Poster K14 - 3D Bioprinting Hepatocytes for In Vitro Liver Tissue Engineering

Thursday, October 24, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Izabela Kowalik, UG

Undergraduate Researcher
National High Magnetic Field Lab
Land O' Lakes, Florida, United States

Co-Author(s)

Nellymar Santiago-Rivera

Undergraduate Researcher
Florida State University/National High Magnetic Field Lab
Tallahassee, Florida, United States
Vivienne Zacher

Undergraduate Researcher
National High Magnetic Field Lab
Hollywood , Florida, United States
Anya Tillman

Undergraduate Researcher
National High Magnetic Field Lab, United States
MS

Mary-Jean Savitsky

Ph.D. Student
National High Magnetic Field Lab, United States

Last Author(s)

JA

Jamel Ali

Assistant Professor
Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Florida, United States

Introduction: Liver disease affects nearly 4.5 million individuals in the United States. Due to the current gaps in knowledge, and in supportive care, about 57,000 of these cases result in mortality. For individuals with chronic liver disease, liver transplantation is many times the only option. However, this solution becomes problematic due to the scarcity of donors and the possibility of transplant rejection. Liver tissue engineering is an evolving field of study that aims to aid in therapeutic and transplantation studies. Within this topic many have turned to the usage of hydrogels as 3D culture systems for tissue models as they hold promise in providing an accurate mimicry of native liver tissue. Here, we aim to understand if liver cells incorporated in alginate hydrogels and a novel micro fibrous matrix can be utilized for 3D bioprinting viable liver tissue models that mimic various hepatic disease states. By characterizing the rheological properties of these hydrogels, we aim to establish a correlation between these properties and their printability, thereby demonstrating their feasibility for 3D bioprinting.

Materials and

Methods: In this investigation, we used 4% (w/v) Alginate hydrogels to act as a mimetic fibrotic liver tissue. Gels were created using BEGM Media and 0.4 g of medium viscosity alginate in a 1:9 ratio with the micro fibrous matrix. Previous work from our group demonstrated that this matrix can promote hepatocyte growth and viability. We subjected fibrous biomaterials encapsulated within hydrogels to a series of bulk rheology tests in to ensure its viscosity was similar to that of a fibrotic liver. Rheological properties were characterized through a series of runs including a frequency sweep, flow curve, and a cross-linked time sweep. Printability was tested on the 4% alginate hydrogels through the changing of many variables using a SunP bioprinter. The main variables that were analyzed in this exploration were print speed, extrusion speed, temperature, and nozzle height. A 5% calcium chloride solution was used for crosslinking prints in between layers. Both droplet and aerosol methods were tested for crosslinking during prints. Prints were measured on a Keyence VHX 7000N microscope, and values were analyzed to identify printability coefficient.

Results, Conclusions, and Discussions: The ability of 4% Alginate gels to flow were verified through rheological flow curves. Additionally, frequency sweeps performed showed consistency between runs and resulted in a near constant storage modulus at a frequency of 1 Hz. We found the best parameters for printability at this alginate percentage to be a print speed of 3mm/s, an extrusion speed of 1.8 mm3/s, temperature of 16°C, and a nozzle height of 1.5 mm from the build platform. An aerosol method of ionic crosslinking provided for an even distribution of calcium chloride over the gels allowing for a better flush print. With this crosslinking method, we were able to achieve a 3D structure of 8 layers before collapsing occurred. Assuming the desired printability coefficient is 1, our value was 0.647. Due to the lack of reliable disease models that are currently available, our goal is to optimize bioinks with the use of a fibrous matrix. Our goal here was to identify the best way to extrude these hydrogels through a nozzle and simultaneously produce 3D structures that can maintain their shape after they had been extruded. An issue we experienced in our studies was that the 5% calcium chloride must be washed off exactly at 3 minutes to obtain the same stiffness of a fibrotic liver. This becomes challenging as it is difficult to wash the gel in between its layers., Future investigations should include the use of a lower percentage calcium chloride to obtain other physiologically relevant liver stiffness values. The bioinks developed in this investigation have the potential of being tuned into viable structures that can accurately mimic the extracellular environment of the liver. We identified a facile method for crosslinking was through aerosol crosslinking which cures the gel evenly after each print. The temperature of the base plate also played a crucial role in the formation of the structure as it allows time to crosslink the hydrogel in between prints.

Acknowledgements (Optional): This work was funded by the National Science Foundation (No. EES-2306449, EES-2219558) and supported by the NSF FAMU CREST Center award (No. EES-1735968). This research work was also supported by The Grainger Foundation Frontiers of Engineering Grant under the National Academy of Sciences Award Number: 200001318. Support was also provided by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R16GM145595. All the work was performed at the National High Magnetic Field Laboratory, which is supported by National Science Foundation Cooperative Agreement No. DMR 1644779 and the State of Florida. Mary Jean Savitsky acknowledges support from the Merk Pathways to Successful Biomedical Careers Graduate Fellowship.