Creation of Trans-Mitochondrial Cybrids

The process of creation of trans-mitochondrial cybrids is described in previous studies [33 (link)]. After collecting 10 mL of peripheral blood using tubes with sodium citrate, DNA extraction kits (Puregene, Qiagen, Valencia, CA, USA) and the Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA) were used for DNA isolation and quantification, respectively. Then, a series of centrifuge steps were performed for platelet isolation followed by using Tris buffer saline (TBS) for platelet suspension. ARPE-19 cell lines, purchased from ATCC (Manassa, VA, USA), demonstrated similar functional and structural characteristics to in vivo RPE cells [35 (link)]. The ARPE-19 were exposed to low-dose ethidium bromide and serially passaged until they were deficient in mtDNA (Rho0) [36 (link)]. As described in previous studies, cybrids were created by fusion of Rho0 ARPE-19 cells with platelets using polyethylene glycol [32 (link)].