研究目的
To compare the cell toxicity of potential pharmacologic alternatives to riboflavin photochemical corneal cross-linking agents.
研究成果
Significant differences in cell toxicity were found among the cross-linking agents, with mononitroalcohols and glyceraldehyde being least toxic, and glutaraldehyde, paraformaldehyde, genipin, and bronopol being most toxic. The balance between cross-linking efficacy and cytotoxicity is crucial for developing safe therapeutic agents. Future studies should explore in vivo applications and optimize compounds to minimize toxicity while maintaining cross-linking effectiveness.
研究不足
The study is limited to in vitro cell culture models, which may not fully replicate in vivo conditions. Variability in cell responses and reagent preparations (e.g., polymerization of glutaraldehyde and paraformaldehyde) could affect results. The toxicity thresholds do not account for differences in reactivity between cell membranes and extracellular matrix proteins. LD50 and genotoxicity data are incomplete for some compounds.
1：Experimental Design and Method Selection:
The study compared nine chemical cross-linking agents using four cell lines to assess toxicity thresholds. The trypan blue exclusion method was employed to determine cell viability after exposure to agents at various concentrations.
2：Sample Selection and Data Sources:
Four cell lines were used: immortalized human corneal epithelial cells, human skin fibroblasts, primary bovine corneal endothelial cells, and immortalized human retinal pigment epithelial cells (ARPE-19). Cells were cultured in specific media under standard conditions.
3：9). Cells were cultured in specific media under standard conditions. List of Experimental Equipment and Materials:
3. List of Experimental Equipment and Materials: Chemicals included 2-nitroethanol, 2-nitro-1-propanol, 2-bromo-2-nitro-1,3-propanediol (bronopol), genipin, L-glyceraldehyde, 2-methyl-2-nitro-1,3-propanediol, 2-hydroxy-methyl-2-nitro-1,3-propanediol, paraformaldehyde, and glutaraldehyde from suppliers like Sigma-Aldrich, Tokyo Chemical Industry, and Electron Microscopy Sciences. Cell culture equipment included 24-well plates, incubators, and an inverted microscope (Fisher Scientific Cat No. 12-560-45).
4：5). Experimental Procedures and Operational Workflow:
4. Experimental Procedures and Operational Workflow: Cells were seeded in 24-well plates, exposed to cross-linking agents at concentrations from 0.001 mM to 10 mM for 24 hours, followed by a 48-hour recovery in fresh media. After recovery, cells were stained with trypan blue for 3 to 60 minutes, washed, and examined under a microscope to assess viability and fixation.
5：001 mM to 10 mM for 24 hours, followed by a 48-hour recovery in fresh media. After recovery, cells were stained with trypan blue for 3 to 60 minutes, washed, and examined under a microscope to assess viability and fixation. Data Analysis Methods:
5. Data Analysis Methods: Toxicity was categorized into five levels (alive, partially dead, dead, dead/gone, dead/fixed) based on morphological observations and trypan blue uptake. Thresholds were defined as the highest concentration where all cells remained alive.
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trypan blue solution
0.4%
Gibco
Used as a vital dye to stain dead cells and assess cell viability.
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epidermal growth factor
EGF
Sigma-Aldrich
Growth factor supplement for cell culture.
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trypsin
0.25%
Gibco
Used to detach cells from culture flasks for subculturing.
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culture flasks
25-cm2
NUNC-Thermo Fisher Scientific
Used for growing and maintaining cell cultures.
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inverted microscope
Cat No. 12-560-45
Fisher Scientific
Used to examine cell morphology and staining characteristics after trypan blue staining.
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24-well cell culture plates
Used for seeding cells and conducting toxicity tests.
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Dulbecco’s modified Eagle’s medium
DMEM
Invitrogen
Culture medium for immortalized human corneal epithelial cells.
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fetal bovine serum
Invitrogen
Supplement for cell culture media.
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insulin
Sigma-Aldrich
Supplement for cell culture media.
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penicillin and streptomycin
Invitrogen
Antibiotic supplement for cell culture to prevent contamination.
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dermal cell basal medium
ATCC
Culture medium for human skin fibroblasts.
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fibroblast growth kit
ATCC
Contains supplements for fibroblast culture, including growth factors and other components.
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Ca2+-free Dulbecco’s Phosphate Buffered Saline
DPBS
Invitrogen
Used for washing cells during culture and toxicity procedures.
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