Two Photon Biofabrication of the Human Lamina Cribrosa for the 3D culture of optic nerve head cells

Abstract

Purpose : The use of two photon polymerization has made the production of nanoscale complex structures like the human lamina cribrosa possible. The production of unique ocular models may prove useful in modeling the mechanobiology of diseases like glaucoma. The purpose of this study is to optimize cell culture conditions for seeding and maintaining optic nerve head (ONH) cells on a fabricated lamina cribrosa (LC) to understand how ONH cells interact with retinal ganglion cells (RGCs).

Methods : For this study we fabricated the human LC by utilizing two photon polymerization on an Olympus BX51 upright laser-scanning microscope (Insight DS+, Spectra Physics, Santa Clara, CA). The laser was centered at 780 nm. The LCs were fabricated out of Nanoscribe’s IP-PDMS, a biocompatible photoresin. After fabrication, the LC was developed and sterilized in 70% ethanol. The cells used in this study were isolated from the LC of porcine ocular tissue post mortem. After isolation, the cells were expanded for 2 passages and then seeded on the structure. The cells were allowed to attach for 1 hour before supplementing the culture vessel with additional medium. Finally, the cells were maintained on the LC for up to 2 weeks at 37°C and 5% CO₂.

Results : Preliminary results demonstrate that the IP-PDMS photoresin is biocompatible. Further, the fabricated structure can sustain ONH cells. When seeded, the cells settle and attach to beams and into the pores of the LC. Figure 1B is an image of the fabricated human LC made using IP-PDMS, and 1A shows the input image used to fabricate the human LC.

Conclusions : While we are able to sustain ONH cells on a fabricated LC, there are limitations to this technique. Our current approach is limited in its ability to promote efficient attachment of cells to the LC. Further work is needed to optimize the attachment efficiency of these ONH cells to the fabricated LC. Finally, a future direction of this project is to understand how these ONH cells contribute to glaucoma development and progression in a co-culture system with human retinal organoid – derived RGCs, utilizing this 3D culture system.
Research reported in this abstract was supported by the NIH T32 Interdisciplinary Visual Sciences Training Program.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Image of a fabricated human lamina cribrosa made using IP-PDMS for the 3D culture of porcine optic nerve head cells (B) and the input image used to fabricate the lamina cribrosa (A).