During vitreoretinal procedures, the visualization of transparent structures such as the vitreous cortex, ERM, and ILM can be a challenge; however, proper staining of these tissues can increase the efficiency of a non-traumatic removal.

Vitreoretinal surgery has rapidly evolved over the past 10 years by the development of new techniques and improvement in vitrectomy machines. Moreover, a big effort has been dedicated to vital dyes formulation to enhance visualization of tissues and facilitate membranes peeling: this leads to a great reduction of complications such as iatrogenic breaks, retinal damages and, eventually, minimal postoperative discomfort for patients.

To guarantee all of this, the dye for the posterior segment of the eye must fulfil some important requirements. First of all, it has to be dense enough to easily lie down and spread onto the membrane without causing any damage because of the excessive weight. It follows that it has to selectively adhere to and stain the posterior segment eye membranes while being soluble in water to be easily washable from the tissues at the end of surgery. Finally, and most importantly, the vital dye must not be toxic for the ocular tissues.

In recent decades has strongly emerged the theme of the safety of vital dyes.

For instance, the indocyanine green has been abandoned, owing to the in vitro and in-vivo toxicity concerns. Instead, the trypan blue-based formulations tailored for use in posterior segment eye surgery are deemed to be safe and are at present unanimously considered the gold standard for ERM surgery.

In our research laboratories, the method of choice to test the safety of the vital dyes for the posterior eye segment surgery has always been a quantitatively and qualitatively validated in vitro cytotoxicity test by direct contact method according to ISO 10993-5.

This testing method has the following advantages:

  • it is highly sensitive and able to detect even weak cytotoxicity;
  • it is performed not only on the mouse fibroblast BALB 3T3 but also the human retina-derived ARPE-19 cell line: in this way, it is possible to meet the recommendations of the ISO standards stating that the in vitro cytotoxicity test method of a product for human use should be selected to represent, as much as possible, the situation in which the product is to be used;
  • although during surgery the dye must remain in contact with the ocular tissues for a very short time (usually few seconds), the cytotoxicity test of the testing product is performed for a contact time as long as 24 hours.


  • Mariotti C. et al. Negative staining of the vitreous with the use of vital dyes. Eur J Pharmacol 2018;28(1):117-118
  • Iuliano L. et al. Idiopathic epiretinal membrane surgery: safety, efficacy and patient-related outcomes. Clin Ophthalmol 2019; 13:1253:1265
  • ISO 10993-5, 2009. Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity. https://www.iso.org/obp/ui/#iso:std:iso:10993:-5:en