Magnetic Targeting of Human Corneal Endothelial Cells

1. Magnetic Targeting of Human Corneal Endothelial Cells José Álvaro Pereira Gomes,MD Gustavo Teixeira Grottone,MD Ana Beatriz Diniz Grisolia, MD Priscila Cristovam,MD Authors have no financial interest  Ophthalmology Department, UNIFESP, São Paulo, SP

2. Purpose • To evaluate morphological changes and response of human corneal endothelial cells exposed to superparamagnetic silicon coated iron oxide particles against magnetic fields.

3. Introduction • Targeting of human corneal endothelial cells is a subject recently studied concerning innovative ways to proceed with cell therapy in cases of endothelial disfunction. • Recent literature showed use of several ways to promote adhesion of these cells to the posterior cornea have been studied. Iron microparticles, prone position of the host and at last superparamagnetic nanoparticles are the most recent effort to achive maximum sucess on cultured HCEC integration at hosts.

4. Methods and Patients • Pre-clinical, in-vitro study. • The corneas used at this study were obtained from Sorocaba Eye Bank/Brazil and were unsuitable for using in human patients because of positive serology for hepatitis virus B or C. • Twelve corneas were dissected under microscope and descemet membrane carrying endothelial cell were incubated overnight with a collagenase A solution ( 1 mg/ml – ROCHE) diluted in DMEM basal media. • Digested material was centrifuged and washed twice in PBS. • Aggregates were resuspended and vigorously pipetted to reduce size of aggregates to provide a better spreading of cells among the uncoated 24-well plates

5. Methods and Patients • 300 nm silicon coated paramagnetic nanoparticles suspension was centrifuged 8 times in PBS to wash any residual preservative at the solution vehicle. • Plates were divided into four groups: • Sham • 0.21mg/ml SPIO • 0.7mg/ml SPIO • 2.1 mg/ml SPIO • Plates were filled with nanoparticles solution in different concentration after cells showed at least 80% confluency. • Filled wells were incubated overnight and supernatant disposed after incubation period.

6. Methods and Patients • Exposed cells were washed three times in PBS solution and incubated with SHEM media for another week • Media was changed every other day. • Cells were harvested by another collagenase A digestion overnight. • Cell aggregates were submitted to magnetic field of 3500 Gauss and movement and velocity was recorded at a Nikon TS-100 inverted microscope with CCD camera attached • Images were processed by NIKON software and estimate velocities were logged.

7. Results • None of the studied samples showed morphological changes after one week after instilation of SPIO nanoparticles • All concentrations tested were able to magnetize all cell aggregates collected after collagenase A digestion(second pass) • Sham group showed no attraction to magnetic stimulus, remaing steady at the duration of tests • Mean displacement velocity among SPIO samples was 15 um/s

10. Conclusions • Morphology of cells sustained the normality between groups which used sham and SPIO in different concentration in short-term. • All tested concentrations of silicon encapsulated SPIOs resulted in consistent and highly efficient magnetization of HCECs when used collagenase method to harvest the exposed cells. • Aggregate formation seems to be superior than single-cell assays because heterogeneous intake of SPIO is partially compensated by clump formation with positive and negative intake cells. Single-cell suspension depends exclusively on positive intake cells.

11. VIDEOProvided as extra file.Cell displacement over time