Master's Student Northwestern University Evanston, Illinois, United States
Introduction: Electrical stimulation of cells is a growing area of interest in the biomedical field. By electrically stimulating cells, intracellular signaling pathways can be activated and cell proliferation and differentiation can be enhanced. The electrical activation of cells has shown great potential for the development of implantable cellular factories that can generate drugs and medications on-demand in the body. One key component of enhancing the therapeutic potential of electro-genetic cells is the development of scaffold-like conductive constructs that can activate cell factories within the bulk material. For this purpose, increasing the adhesiveness of cells onto selected biomaterials is essential. High cell adhesion to the scaffold allows successful cell stimulation and subsequent cellular activities, which facilitate the integration of the bioelectronic platform into the body. To enhance the conductive scaffold adhesiveness, fibronectin was utilized for the following experiment. Fibronectin allows the construct to mimic the extracellular matrix and enhances the strength and duration of cell adhesion to constructs. In this specific abstract, we aim to investigate the effect of fibronectin on cell viability on selected biomaterials, specifically, graphene foams, platinum (Pt) meshes, and Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) particles. All materials are known for their biocompatible properties. By examining the cell viability after adding fibronectin, we seek to determine whether these constructs are still biocompatible and practical for biomedical applications. This study expects to demonstrate that the addition of fibronectin to graphene, Pt meshes, and PEDOT enhances cell adhesion without affecting cell viability, making these materials more suitable for developing effective bioelectronic platforms.
Materials and
Methods: The experiment was done in 3 steps: seeding the cells, culturing the cells, and analyzing the cell viability. ARPE19 cells, which are retinal epithelial cells, were prepared for the cell seeding. Before the experiment, we cut each graphene, Pt mesh, and PEDOT to fit in the 10x10 well and sterilized using ethanol. For fibronectin coating samples, we prepared 10 mg of fibronectin and diluted it with 230 µL of PBS. We coated 6 wells with 40 µL of the prepared fibronectin and the remaining 6 wells with 40 µL of DI water for the control samples. After incubating for 45 minutes at room temperature, we removed the rest of the solution and seeded 20,000 ARPE19 cells per well. Afterwards, we added 200 µL of cell media DMEM and incubated the cells. Once in two days, the media was substituted by the 200 µL of the new media.
After 7 days of initial seeding of cells, we analyzed the viability and cytotoxicity of the fibronectin-coated and uncoated cells using Calcein AM and Ethidium homodimer-1(EthD-1). The staining solution was created by mixing 5 µL of Calcein AM with 20 µL of EthD-1 and 10 mL of PBS solution. After removing the medium from the cells, we added 100 µL of the mixed staining solution in each of the wells and incubated for 30 minutes protected from light with aluminum foil. We then analyzed the viability by capturing the image and counting the number of dead/live using Fiji.
Results, Conclusions, and Discussions: Figure 1 displays images of cells stained with Calcein AM and EthD-1. Cells on graphene formed web-like structures, while cells on Pt mesh were predominantly located at the edges and exhibited a stretched morphology. The image of adhered cells for the PEDOT particles was less visible than the other materials, due to the dark-colored and porous nature of the PEDOT particles. However, as observed in the images, all samples demonstrated high cell viability, with green-stained (live) cells vastly outnumbering red-stained (dead) cells. Quantitative analysis of live and dead cells, conducted using Fiji and presented in Figure 2, indicates that platinum mesh and PEDOT samples exhibited relatively high cell viability, each averaging 99% and 100%, regardless of fibronectin coating. In contrast, graphene samples showed slightly lower viability, averaging 87%. As the experiment used small graphene foams with a relatively high surface area to volume ratio, this high surface reactivity might have generated oxidative stress to induce cell death.
Revisiting the purpose of the experiment, we found that the addition of fibronectin coating did not significantly affect the cell viability of any type of scaffold constructs. There was no observed increase or decrease in cell viability. However, if fibronectin indeed enhances cell adhesion compared to samples without fibronectin, it would be a promising method for improving the biocompatibility of conductive scaffolds and the efficiency of cell stimulation in producing the desired therapeutic agent. Additionally, platinum mesh and PEDOT demonstrated higher cell viability than graphene. Therefore, if prioritizing cell survival, platinum mesh and PEDOT would be the preferred base scaffold material over graphene. For future studies, we aim to investigate whether adding fibronectin enhances the secretion of the desired therapeutic agent via activating cell stimulation and to evaluate which type of scaffold achieves the highest activation.
