Perfluoro-n-octane cytotoxicity in human retinal pigment epithelial cell culture

Abstract

Purpose : Several cases of acute blindness post non – complicated retinal detachment surgery produce recently a sanitary alert in Spain attributed to several lots of perfluoro-n-octane (Alaocta® PFO) from Ala Medics®. These lots were certified non-toxic by performing cytotoxicity test of extracts of PFO on fibroblasts cultures following UNE EN ISO 10993 - 5 & 12 : 2009 norms by a certified sanitary product testing company. This study proposes to compare this extract method to a new direct exposure method (in patent consideration).

Methods : The cultures of ARPE19 (retinal pigment epithelial cells) and L929 cell line (fibroblasts) were grown at a seeding density of 1 x 10⁵ to reach an OD ≥ 0.2. The extracts were prepared by stirring 0.2 g/ml of PFO samples in DMEM culture medium for 24 hours at 37 ⁰C. Cell cultures were incubated with these extracts for 72 hours in standard culture conditions followed by XTT assay to detect cytototoxicity. Cultures were also exposed directly to PFO, blocking its evaporation by cell culture medium, for 30 and 60 minutes. Cultures were grown for 24 and 72 hours and cytototoxicity were detected by MTT assay. Experiments were performed fulfilling the requirements of the ISO 10993 - 5 & 12 : 2009 norms and repeated three times in triplicate (n=9).

Results : Both cultures (ARPE19 and L929) grew well and achieved standards required in ISO norms. The experiments performed for extract method showed non-toxic nature of PFO (> 70% viability; non-toxic; < 70%; toxic according to ISO norms). However, the new proposed direct method is capable to differentiate between non toxic and toxic PFO lots. Some lots were more toxic and showed severe damages in cell morphology and cell cultures and related to the permanence time of PFO in storage.

Conclusions : Ala Medics® PFO time-dependent cytotoxicity has been confirmed in ARPE19 cell cultures. This study shows that extract preparation, and testing extract with cultures does not guarantee to detect a toxic component if it is non leachable. Parameters of a test are critical to certify a sanitary product.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.