Targeted microfluidic manufacturing for functionalized biomaterials: Encapsulation of neural cells into conductive alginate microfibers
Three-dimensional cell culturing techniques continue to grow in importance as researchers strive for physiological relevancy in their experiments. In particular, neuroscience benefits from technological advances in this field, as it helps to elucidate the inner workings of this utterly important and yet fundamentally complex organ, the brain and the blood-brain barrier. Current methodologies are well suited for studying the macroenvironment of the brain, but studying thebehavior of cells on a small-scale or single-cell level remains difficult. This work addresses the technological gap by providing innovations for the creation of three-dimensional microfibrous cell culturing platforms, which are capable of encapsulating cells into a system that is more physiologically relevant than the prevailing two-dimensional cell culturing techniques.
Such a platform enables long-term observation of small cultures of cells by entrapping them within the boundaries of hydrogel microfibers. The fibers are fabricated with a cutting-edge technique, which utilizes a microfluidic device to ensure that a pre-polymer solution might flow adjacent to other fluids without turbulent mixing. The pre-polymer solution is gelled by ionic cross-linking with the sheath fluid, which also guides it through the channel, preventing clogging and providing hydrodynamic focusing that gives a high degree of control over the fiber size and shape. Making modifications to the device design creates ideal conditions for fabricating a wide array of fibers with different sizes, shapes, and geometries, allowing for the modeling of a wide range of tissues. This work uses microfluidic manufacturing methods to the creation of alginate-based fibers, both solid and hollow, and explores how they might be combined with conductive graphene solutions and applied to problems within the field of neural tissue modeling.
Initially, optimization of the manufacturing parameters involved with microfluidic fiber fabrication was carried out. The viscosities of the solutions and the flow rate ratios between the involved fluids in the microfluidic device were studied with respect to the surface topography and roughness, thereby providing insight into the hydrodynamic focusing and the relative degrees of turbulent movement between the fluids travelling through the microfluidic device. By manipulating these factors, fibers with average surface roughnesses (Ras) from 1.294 ± 0.324 to 3.100 ± 0.580 were fabricated. Additionally, the effect of cross-linking density on the mechanical properties of the fibers and the survival of encapsulated cells were analyzed, both by changing the concentrations of the cross-linking agents (calcium chloride dihydrate) in the sheath fluid and in the collection bath. Microfibers with a wide range of Young’s modulus (400 to 17,000 MPa) and porosities (12% to 92%) were fabricated. It was found that more crosslinking correlated to higher cell viability; however, flow rate more greatly influenced the fate of encapsulated cells. Cells encapsulated with higher flow rates that spent less time within the microfluidic device had a significantly higher initial viability than cells encapsulated with slower flow rates.
Once the fabrication platform was better understood and characterized, work proceeded by integrating aqueous graphene solutions into the alginate solutions before manufacturing to ensure that the hydrogels have physiologically relevant conductivities to the native brain tissues, and to work towards future applications wherein the hydrogels can act as real-time sensing mechanisms for cell-to-cell communications. To further understand the implications of including graphene into the fibers, RT-qPCR was utilized to study the prevalence of key neural genes. This was done both after manufacturing and after prolonged encapsulation to understand the genetic effects of the manufacturing process itself compared against the effects of encapsulation and long-term contact with alginate and graphene. Genetic results showed that the manufacturing process and contact with graphene leads to short-term upregulation of TH and downregulation of TUBB-3, indicating increased amounts of dopaminergic neuronal markers and decreased levels of neurogenesis, axonal growth, and maintenance. Long-term encapsulation maintains increased TH and decreased TUBB3 levels, but long exposure to graphene causes a sharp upregulation of TUBB-3, indicating increased amounts of neurogenesis. Likewise, extended encapsulation or contact with graphene causes upregulation of TNF-α or IL-1β, respectively, indicating inflammation.
Hollow microfibers are also of interest in neural studies, since they are well suited to model the blood-brain barrier (BBB). The microfluidic microfiber manufacturing technique can be readily applied towards this goal by utilizing a five-inlet microfluidic device, which enables the creation of a hollow channel through the microfiber. Neural cells were encapsulated into hollow alginate microfibers, and their viability and genetic responses were studied to understand the effect of the manufacturing procedure. Cells maintained approximately 60% viability throughout the three-day observation period, but qPCR showed that one day after encapsulation, cells showed lower amounts of neurogenesis than their non-encapsulated counterparts. The hollow fiber fabrication process and its parameters needs to be optimized to more closely mimic the BBB, while reducing the inflammation of the encapsulated cells.
Overall, the three-dimensional cell culturing technique, with the aid of the microfluidic manufacturing approach, is a functional method to engineer targeted cell scaffoldings to mimic native tissues within the body, such as the brain. The work described in this thesis entails methods used to reliably encapsulate cells within the body of the microfibers for long-term spatiotemporal control and observation, enhance their electrical properties with graphene, and utilize multiple microfluidic devices to fabricate fibers with different cross-sections to mimic both the structure of the brain and the BBB. The genetic responses of the cells were studied after manufacturing and after long-term encapsulation to understand cell health and behavior. These tasks were accomplished to create state-of-the-art, targeted biomaterials for use in modeling neural tissues.