Suspended extrusion-based bioprinting to establish a 3D in vitro model of a neural network to study Parkinson’s disease

Abstract

Parkinson’s disease is a common disorder that affects a rapidly increasing number of people worldwide. In Parkinson’s disease, cells of the central nervous system stop working or even die, resulting tremors, stiffness, balancing issues, rigidity, and many other problems. The disease usually starts in adulthood and progresses over time, but no cure has been found yet. Therefore, it is important to study Parkinson’s to reveal the causes and effects of the disease. Traditionally, we do this in simplified 2D models, which do not properly mimic the 3D environment of cells in the brain, or in animal models, which do not consist of human cells, meaning they cannot properly replicate human diseases. Therefore, in this project, we tried to print a small 3D piece of human brain tissue, as a more life-like prototype version of a disease model that we can use to study Parkinson’s in the future. 3D bioprinting is a relatively new method in medicine to develop human tissue by extruding inks consisting of living cells, called bioinks, in a desired architecture. After the cells are printed, they can proliferate, specialize and mature to form a wide variety of tissues, including bone and cartilage. This tissue can be used for implantation into the body after injury or disease or it can be used in the lab to study disease mechanisms, as in our case.

First, we developed and characterized a support bath consisting of modified gelatin into which a bioink could be printed to prevent the collapse of the printed construct and to keep the cells of the construct in the right place. Since the brain consists of a high number of neurons in a relatively fluid environment, we replicated this by using soft, water-rich materials. Then, we printed with three different bioinks, consisting of cells and additional material to encapsulate the cells for protection and to make the ink flow smoothly out of the cartridge. Because cells generally do not like the printing process and easily get stressed or harmed, sturdy cells from the connective tissue were used to set-up and improve the printing process in the first part of the project. When that was established, embryonic stem cells were used in the second part of the project. They functioned as a precursor for the desired cells making up the construct in the third and last part of the project: neural cells. In this part, we added additional materials to the support bath and bioink to which the cells could bind to stimulate the formation of extensions of their cell body. These extensions are nerve fibers, which are essential for communication between neurons and the establishment of a well-functioning network that allows our central nervous system to perform actions.

In this project, we showed that the newly developed support bath was suitable for 3D printing of all three cell types and that they survived the printing process well. We can conclude that neural cells bioprinted into the support bath that was reinforced with additional materials, were able to specialize and become neurons. They formed many nerve fibers extending out to each other in multiple directions to form a 3D network. These promising results support a new strategy for printing soft constructs, which may help researchers worldwide to improve disease models, including those for Parkinson’s. These may be applied in research, for drug-testing, and personalized medicine in the future.