3D cell scaffolds with enhanced conductivity can offer numerous advantages in creating physiologically relevant platforms in vitro, one of which is the brain tissue with integrated real time sensing. The Hashemi Lab, led by mechanical engineering (ME) associate professor Nichole Hashemi, has developed a high throughput 3D biofabrication technique to encapsulate cells in highly tunable alginate and graphene alginate fibers. The team has developed conductive microstructures with encapsulated neural cells to mimic the micro-tissue environment present in the blood brain barrier (BBB).
The microfluidic cell encapsulation in the alginate and graphene-alginate based microfibers and its short and long-term effects on the gene expressions of the encapsulated cells is described in an article published in Advanced Biology. In addition to Hashemi, the research team includes first authors Marilyn McNamara, a recent doctoral graduate from the Hashemi lab, and Saurabh Aykar a doctoral student in mechanical engineering; Nima Alimoradi a doctoral student in ME; Amir Niaraki, Rajeendra Pemathilaka, and Alex Wrede, also doctoral graduates from the Hashemi lab; and Reza Montazami, an associate professor of ME.
This technique employs a microfluidic method through which two different biocompatible polymer solutions, polyethylene glycol, and alginate/graphene-alginate (Alg/G-Alg) were introduced at a desired flow rate ratio to obtain the alginate or graphene-alginate microfibers. The hydrodynamic shaping within the microfluidic chip shapes the two solutions, with Alg/G-Alg solution containing cells, to get a desired fluid regime. Subsequently, the ionic cross-linking between the alginate and calcium ions from the calcium chloride bath solution polymerizes the Alg/G-Alg to obtain the cell-encapsulated continuous microfibers. The conductivity of the G-Alg microfibers was measured to be 148 % higher than the Alg microfibers which is similar to the conductivity of the native brain tissue. Moreover, the cell encapsulation technique had an efficiency of 50% and 30% of those survive for a total of 6 days post manufacturing. One of the advantages of this technique is the simultaneous encapsulation of cells in the microfibers while manufacturing. Furthermore, the highly porous fibers enhanced the diffusion potential for waste and nutrients to and fro from the fiber thereby potentially influencing cell viability and gene expressions.
The study entails the effect of the microfluidic encapsulation process on the encapsulated cells for both short and long term, by analyzing four genes, tyrosine hydroxylase (TH), tubulin beta 3 class 3 (TUBB3), interleukin 1 beta (IL1β), and tumor necrosis factor alfa (TNFα) that are important to neural health. To monitor the health of the dopaminergic neural cells, a rate limiting enzyme in dopamine synthesis TH, and TUBB3 which is responsible for forming cellular cytoskeleton that aids neurogenesis, organellar transport, and cell migration were evaluated using reverse transcription polymerase chain reaction and enzyme linked immunosorbent assay. Moreover, proteins IL1β and TNFα were assayed to identify the potential inflammatory response within the neural cells that is caused by increase in the microglia activation. These proteins were evaluated both immediately after manufacturing the cell encapsulated microfibers and after prolonged encapsulation.
A similar upregulation of TH level was observed on day 1 in both Alg control (cells in Alg precursor solution) and Alg fiber as compared to control indicating that the manufacturing process possessed no effect on the TH expression. Furthermore, the G-Alg fibers had a higher TH expression level as compared to Alg fiber suggesting that the contact with graphene increased the expression of TH. In case of TUBB3, a similar level of downregulation was observed in both Alg control and Alg fiber proving no manufacturing effect on the TUBB3 expression level. Additionally, the contact of graphene caused more downregulation of the TUBB3. Since the expression levels of TUBB3 were less than the lower detection limit of ELISA, it can be considered that the cell encapsulation process had no adverse effects.
A similar trend was observed on day 6 for both TH and TUBB3 indicating that the long-term culture possessed little to no change in the expression of these genes as compared to day 1. In case of IL1β, a downregulation was observed in both the Alg control and Alg fiber as compared to the control during the long-term culture. However, an upregulation was observed in the G-Alg fiber indicating that the prolonged contact with graphene triggered the inflammatory response of the neural cells. An upregulation was observed in the expression level of TNFα for Alg fibers as compared to the Alg control after the cells were in contact with the alginate for a prolonged period. However, a downregulation was observed in the G-Alg fibers indicating the prolonged contact with graphene reduced the inflammatory response of neural cells.
Other cell encapsulation techniques such as electrospinning or extrusion possess a limit on the lower dimension of the fiber manufactured. Moreover, the cells manufactured by these methods undergo adverse manufacturing stresses and require some post-processing techniques. However, cell encapsulation using the microfluidic approach bears minimal manufacturing effects on the encapsulated cells and is free from any post-processing techniques.
The fibers can be made biodegradable or retrievable depending on the type of its application. The cell encapsulated in the biodegradable fibers can potentially be used to treat micro-tissue areas that lack either a specific cell type or desired cell population. Moreover, this graphene incorporated fibers can be used in conjunction with 3D micro-electrode arrays to have a real time sensing ability for analyzing cell-to-cell interaction occurring within the brain tissue.
The work currently supported by the National Science Foundation STTR grant proves the feasibility and application of this BBB technology to streamline drug development for many different industries. Although the main focus is human applications, due to the interchangeable cell design, this model can also be utilized for animal applications to aid in Iowa’s vast livestock agriculture businesses. Pharmaceutical companies located around central Iowa such as Merck have acknowledged the value of this emerging technology for drug and vaccine development.