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D. J. McCanna, A. K. Weeks, M. B. Gorbet; Evaluation of the Viability of THP-1 Derived Macrophages Using Ethidium Homodimer-1, Calcein, and AlamarBlue to Determine the Biocompatibility of Intraocular Lens Materials. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4574.
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© ARVO (1962-2015); The Authors (2016-present)
Macrophage adhesion to intraocular lens (IOL) materials may play a role in the biocompatibility of IOL. An in vitro model was developed using silicone and novel IOL materials to evaluate the viability of THP-1 derived macrophages on the materials and after exposure to various concentrations of zinc diethyldithiocarbamate (ZDEC) and benzalkonium chloride (BAK).
THP-1 derived macrophages were cultured onto HEMA and silicone hydrogels containing crosslinked hyaluronic acid (HA) and to a silicone film control biomaterial deposited with known inflammatory chemical ZDEC (0.01%-0.1%). After incubation in RPMI medium with 10% serum, adhered macrophages were evaluated for viability. Macrophages were also exposed to BAK at 0.001 to 0.1%. Using confocal microscopy, the number of live and dead cells adherent to the biomaterial was determined by imaging for calcein and ethidium homodimer-1(EthD-1) fluorescence. The cell metabolic activity was determined using alamarBlue. The degree of damage to cellular membranes was assessed using EthD-1 and the LDH assay.
The adhered cells to the ZDEC containing biomaterial stained for EthD-1 indicating that all of the attached cells had damaged cell membranes. The majority of the cells attached to the silicone control material without ZDEC were live cells (calcein-stained cells). Cells on the HEMA and silicone hydrogels containing HA all stained with calcein, however different numbers of macrophages were observed. The degree of adhesion appeared to be influenced by the molecular weight of HA. As expected, the ZDEC containing biomaterial and BAK caused a decrease the metabolic activity (p < 0.05). Compared to the EthD-1 assay and the alamarBlue test, the LDH assay was not effective in showing cell membrane damage as no dose relationship was observed.
This evaluation demonstrates that the viability of THP-1 derived macrophages adhered to biomaterials can reliably be assessed with the fluorescent dyes EthD-1, calcein and alamarBlue. Further investigations are underway to assess how HA-containing hydrogels may improve IOL biocompatibility by reducing interactions with macrophage.
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