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Vivek Shah, William W Li, Michael Molyneaux; Growth factor characterization of living human activated leukocyte suspension (LHALS) and effect on primary human corneal epithelial cell proliferation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):709.
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© ARVO (1962-2015); The Authors (2016-present)
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).
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.
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).
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.
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