Poster J20 - An Optical Interface to Examine Electrotaxis in Eye Facsimiles

Thursday, October 24, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Sakshi Koul (she/her/hers)

Incoming PhD Candidate in Biomedical Engineering
Rutgers University, New Brunswick
New Brunswick, New Jersey, United States
Luke Devecka

Undergraduate at Rutgers University
Rutgers University, New Brunswick
Morganville, New Jersey, United States

Co-Author(s)

MP

Mark Pierce, PhD

Professor of Biomedical Engineering
Rutgers, The State University of New Jersey, United States
Maribel Vazquez, PhD (she/her/hers)

Professor of Biomedical Engineering
Rutgers, The State University of New Jersey
Piscataway, New Jersey, United States

Introduction: Dysfunction in the retina leads to vision loss in mature and aging adults, which will affect an unprecedented 500 million people by 2050. Regenerative medicine offers the exciting promise to restore vision through cell replacement therapy, where stem and progenitor-like cells (SCs) are transplanted into the retinal host to replace damaged and/or degenerated neurons in the retina. In vivo, SCs must migrate into the retina to replace damaged neurons. Migration of SCs have been observed under electric field in microfluidic channels and other devices like the microeye previously developed by our group. However, there still a lack of studies on the effects of electrotaxis of SCs into the retina, especially under variable EF. This project explores the use of electric fields (EF) to stimulate SC movement within eye facsimiles made of common biomaterials. We present a novel device called the “Parallel Eye” to apply EF across retinal progenitor cells within 3D eye facsimiles of collagen, gelatin, and alginate. Our device is compatible with confocal microscopy to validate cell viability and measure electrotaxis without need of cryosectioning.

Materials and

Methods: The Parallel Eye has four cylindrical chambers of 1-cm-diameter and 3-cm-length (Figure 1A and 1B) fabricated by 3D printing with Nylon X carbon fiber. The Parallel Eye was filled with alginate hydrogel to form the alginate-cell-facsimile resistor model (Figure 1C), and assembled as in Figure 2. The EF was calculated by measuring the potential drop ΔV applied across the length of the alginate-cell-facsimile resistor L using E=ΔV/L. Three types of alginate eye facsimiles were made using de-ionized (DI) water, serum free Dulbecco’s Modified Eagle Medium (DMEM) and Dulbecco’s Phosphate Buffered Saline (DPBS) separately, the latter two to ensure cell viability for future testing using conventional protocols. Collagen IV hydrogel and Gelatin hydrogel were also made using conventional protocols. lThe potential drop across the Parallel Eye was tested in the three types of alginate hydrogel along with Collagen IV and Gelatin hydrogels. Eye facsimiles were seeded with R28 cells by mixing a DMEM cell-suspension at 2×106 cells/mL with a 5% w/v sodium alginate solution in a 1:3 ratio. The facsimiles were stained with Hoechst in DMEM at 1µL/mL and incubated for 30 minutes or stained with proflavine by submerging eye facsimiles in 0.01% w/v proflavine in PBS and incubated for 5 minutes. After staining, the cells were imaged at defined focal planes within intact facsimiles using confocal fluorescence microscopy.

Results, Conclusions, and Discussions: Results and

Discussion: Simulation of electric fields and potential migration of cells was modeled in COMSOL. A heatmap across the cell layer and facsimile shows that the electric field across them remains relatively constant, as shown in Figure 3. A potential drop was observed across all three facsimiles of alginate, collagen IV, and gelatin, and their corresponding EF were averaged across all four chambers as shown in Figure 4. We were also able to show on confocal microscopy the presence of live cells in the eye facsimile after an electric field of 100 mV/cm was applied for five minutes and then incubated for 24 hours using Hoechst staining for live cells or proflavine staining for total cell count at a height of 0.23mm from the bottom of the 1.05mm diameter facsimile as shown in Figure 5.

Conclusion: We have demonstrated a new device that enables the study of cell migration in 3D eye facsimiles under consistent applied electric fields. Device advantages include excellent cell-viability, ease of production, and cost-effectiveness via 3D printing using carbon fiber. In addition, the device uses the resistor model for creating EF, which enables electrotaxis testing within eye facsimiles under variable electric fields/potential drops in the future. Most significantly, the system facilitates imaging of cell migration within the intact facsimile via confocal microscopy, eliminating specialized and time consuming cryo-sectioning of samples.

Acknowledgements (Optional):