Microfluidic Cell Sorting for Autologous Photoreceptor Replacement Therapies

Abstract

Purpose : Enrichment of photoreceptor cells from organoids with complex cellular composition is an important step in the development and optimization of photoreceptor replacement therapies. In this study, we present a microfluidic cell sorting platform for isolating photoreceptor cells from retinal organoids differentiated from patient derived induced pluripotent stem cells (iPSCs) to enable the manufacture of cell therapies with defined cellular composition under current good manufacturing practices (cGMP).

Methods : PDMS microfluidic channels were cast on Silicon/SU-8 molds and bonded to glass. To evaluate the effect of pressure and channel geometry on device performance, fluorescent microparticles with diameters comparable to retinal cells were used. Fluorescent reporter lines were generated to enable optimization of retinal cell sorting.

Results : To determine whether the microfluidic sorting device would be effective for particles in the size range of retinal cells, we pumped suspensions of fluorescent microparticles with two different diameters (6μm and 13μm) through a single microchannel at two different pressures (Figure 1). While 6μm particles are scattered across the channel width at both 100mbar and 400mbar input pressure, larger 13μm particles focused into a narrow stream at 400mbar, implying that proper arrangement of outlets could yield samples enriched for both particle populations. Reporter constructs designed to label specific populations of retinal neurons were developed and confirmed to drive expression of a fluorescent reporter in their respective cell types (i.e., RHO-GFP, Gnat2-YFP, BEST1-mCherry, mGluR6-BFP, BRN3-RFP). Following validation, stable iPSC reporter lines were generated and differentiated into retinal organoids to enable optimization of microfluidic cell sorting.

Conclusions : In this work we present initial findings which suggest that microfluidic cell sorting could be used to isolate smaller photoreceptor cells desired for transplant from other larger cell types such as glial cells present in patient iPSC derived retinal organoids. To enable the optimization of channels for sorting retinal cells we have developed iPSC lines which express several fluorescent reporters under the control of promotors unique to the various cell types present in retinal organoids.

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

 

Figure 1: Selective focusing of fluorescent microspheres. Brighter regions of the image correspond to greater particle density.