Abstract
Purpose :
The purpose of this study is to develop a novel culture system capable of providing tightly controlled uniaxial flow to cells cultured on the bottom of a transwell insert to replicate the mechanical stresses applied to choroidal endothelial cells in vivo. In addition to providing a platform capable of elucidating the connection between applied fluidic shear with the molecular and morphological maturity of endothelial cells, by placing patient-matched iPSC-derived retinal pigment epithelium (RPE) and fully differentiated iPSC-derived neural retina on top of the transwell, this system will constitute an in vitro model of the outer human retina.
Methods :
Our disposable, single use transwell perfusion system was printed using an Object30 Prime 3D printer (Stratasys). After printing the device, cells from a human choroidal endothelial cell line were seeded on transwell inserts, after which the culture system was assembled by placing the transwell inserts into the device, which in turn was placed in a six well plate. A peristaltic pump was then used to provide controlled fluid flow across the seeded endothelial cells. After the conclusion of a perfusion experiment, the transwell membrane can be recovered from the system, allowing observation of cell morphology via microscopy as well as analysis of the molecular characteristics of the perfused cells.
Results :
As per Figure 1, the perfusion device was designed with rectangular flow channels to induce uniaxial flow. Cells perfused using this system remained viable for 72 hours post-perfusion (417µl/hour) and remained attached to the surface of the transwell insert following removal from the perfusion device. Now that we have established that the system can apply controllable fluidic shear stress for days while keeping perfused cells viable, we will proceed to investigate the effect of increasing rates of shear stress on the morphological, molecular, and functional maturity of choroidal endothelial cells with and without RPE plated above.
Conclusions :
We have used modern rapid-prototyping techniques to develop a perfusion system capable of testing the effect of fluidic shear on the phenotype and behavior of choroidal endothelial cells in vitro. Further, we believe that this system can be extended to serve as an in vitro model of the entire retina and used to determine the causes of and evaluate treatments for inherited blindness.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.