Abstract
Purpose:
Corneal ulceration can result in loss of vision and severe discomfort for patients. Current treatment for these corneal ulcers/wounds include topical cycloplegics, conjunctival grafts, and corneal transplants. We developed a living human activated leukocyte suspension (CureXcell™; LHALS) that is currently in Phase III development for wound healing. This study characterized and determined the effect of LHALS on human primary corneal epithelial cells (HCEC).
Methods:
The concentration of EGF, HGF, NGF, BDNF, VEGF and PDGF-BB present in LHALS vs inactive leukocyte suspension was determined by Luminex assay (n=3 batches). Cell proliferation assays (n=4/group) was performed on HCEC: (i) co-cultured with 4µm trans-well inserts containing LHALS, (ii) cultured in control cell growth media, and (iii) cultured in rhEGF. After 48 incubation, HCEC proliferation was measured by an (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) MTS colorimetric assay. Two-tailed paired t-test was used for Luminex and one-way ANOVA with tukey post-hoc was used for cell proliferation.
Results:
Luminex analysis demonstrated that LHALS contained significantly higher amount of EGF (57±18 pg/ml; p=0.037), HGF (1245±368 pg/ml; p=0.031), NGF (22±3 pg/ml; p=0.003), BDNF (3217±103 pg/ml; p<0.001), VEGF (1219±310 pg/ml; p=0.008) and PDGF-BB (397±69 pg/ml; p=0.005) compared to the inactive leukocyte suspension (2±0 pg/ml, 160±39 pg/ml, 8±1 pg/ml, 213±27 pg/ml, 22±6 pg/ml and 77±10 pg/ml; respectively). HCEC treated with LHALS exhibited significantly increased cell proliferation (OD value 2.23±0.38; p=0.0247) compared to control (OD value 0.18±0.02). On an average, LHALS-treated cells reported higher cell proliferation than rhEGF treated cells (OD value 1.64±0.47; p=0.562).
Conclusions:
LHALS technology represents a potential novel modality for accelerating corneal repair. In-vitro studies demonstrated LHALS: (i) contains biochemical factors that play a role in corneal repair; and (ii) enhanced HCEC proliferation by 12-fold as compared to the control. Further studies are underway to delineate the mechanism of action and examine the effect of LHALS in in-vivo models of corneal wound healing.