Limits...
In vitro growth of lens epithelial cells from cataract patients - association with possible risk factors for posterior capsule opacification.

Sundelin K, Petersen A, Soltanpour Y, Zetterberg M - Open Ophthalmol J (2014)

Bottom Line: Lower age and female sex showed border-line significant associations with a higher rate of cell proliferation.The presence of pseudoexfoliation in vivo did not significantly affect cell growth in culture postoperatively.The lack of strong correlations between in vitro growth and known risk factors for PCO in the donors suggest that other factors than the proliferative capacity of the cells per se are important for PCO formation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience and Physiology, Section of Clinical Neuroscience and Rehabilitation/Ophthalmology.

ABSTRACT

Aim: Inter-individual differences in intrinsic proliferative capacity of lens epithelial cells may have importance for the risk of developing posterior capsule opacification (PCO) after cataract surgery. The purpose of the present study was to determine growth of human lens epithelial cells (HLEC) in culture and investigate possible associations with clinical characteristics of the donors, such as age, sex, pseudoexfoliation, uveitis and diabetes.

Methods: Pieces of lens capsule and adhering lens epithelial cells were obtained through capsulorhexis at cataract surgery. Specimens were cultured in a humidified CO2-incubator using standard culture medium and 5% fetal calf serum for two weeks after which cultured cells were stained with carboxy-fluorescein diacetate succinimidyl ester. Image processing software was used to determine the area of the confluent epithelial cell layer in relation to the size of the original capsule specimen.

Results: The increase in area of confluent HLEC showed a negative correlation with diabetes at the first week after surgery. Lower age and female sex showed border-line significant associations with a higher rate of cell proliferation. The presence of pseudoexfoliation in vivo did not significantly affect cell growth in culture postoperatively. Nor did installation of xylocain in the anterior chamber during surgery.

Conclusion: Diabetes is associated with lower rate of proliferation of lens epithelial cells in culture. The lack of strong correlations between in vitro growth and known risk factors for PCO in the donors suggest that other factors than the proliferative capacity of the cells per se are important for PCO formation.

No MeSH data available.


Related in: MedlinePlus

Human lens capsule epithelium specimens after 1 and 2 weeks of culturing respectively.Human lens capsule epithelium specimens were obtained at cataract surgery. Figs. (1A, B) show the same specimen, derived from an 85-year-old woman, after 1 week (A) and 2 weeks (B) respectively. The size of the capsule before outgrowth of epithelial cells was 5 mm. Lens epithelium cells were stained with the non-toxic fluorogenic compound carboxy-fluorescein diacetate succinimidyl ester (CFDA SE) to facilitate monitoring of cell growth. Original magnification x20 is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4066363&req=5

Figure 1: Human lens capsule epithelium specimens after 1 and 2 weeks of culturing respectively.Human lens capsule epithelium specimens were obtained at cataract surgery. Figs. (1A, B) show the same specimen, derived from an 85-year-old woman, after 1 week (A) and 2 weeks (B) respectively. The size of the capsule before outgrowth of epithelial cells was 5 mm. Lens epithelium cells were stained with the non-toxic fluorogenic compound carboxy-fluorescein diacetate succinimidyl ester (CFDA SE) to facilitate monitoring of cell growth. Original magnification x20 is shown.

Mentions: After one week, cultured cells were incubated with carboxy-fluorescein diacetate succinimidyl ester (CFDA SE) for 15 minutes at room temperature. CFDA SE (Vybrant CFDA SE, Invitrogen, Eugene, OR, USA) is a fluorogenic compound that, upon entering metabolically active cells, makes them fluorescent (excitation 492 nm, emission 517 nm). Being non-toxic to the cells, CFDA SE does not affect further growth of the cells, enabling continuous monitoring of proliferation. One week after the first staining procedure, however, the fluorescence had faded and cells were again incubated with CFDA SE. Prior to staining, a stock solution of 10 mM CFDA SE was prepared in dimethyl sulfoxide (DMSO). This stock solution was then further diluted to 10 µM in preheated phosphate buffered saline (PBS). After 15 minutes the CFDA SE solution was replaced by freshly prepared cell culture medium with 5% FCS and lens capsule epithelium specimens were viewed and photographed in a confocal microscope (Nikon Eclipse TE300 C1) using a Nikon D-Eclipse C1 camera and EZ-C1 3.30 Gold as imaging software. Fig. (1) shows an example of a capsulorhexis specimen after staining with CFDA SE after 1 and 2 weeks of culture respectively.


In vitro growth of lens epithelial cells from cataract patients - association with possible risk factors for posterior capsule opacification.

Sundelin K, Petersen A, Soltanpour Y, Zetterberg M - Open Ophthalmol J (2014)

Human lens capsule epithelium specimens after 1 and 2 weeks of culturing respectively.Human lens capsule epithelium specimens were obtained at cataract surgery. Figs. (1A, B) show the same specimen, derived from an 85-year-old woman, after 1 week (A) and 2 weeks (B) respectively. The size of the capsule before outgrowth of epithelial cells was 5 mm. Lens epithelium cells were stained with the non-toxic fluorogenic compound carboxy-fluorescein diacetate succinimidyl ester (CFDA SE) to facilitate monitoring of cell growth. Original magnification x20 is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066363&req=5

Figure 1: Human lens capsule epithelium specimens after 1 and 2 weeks of culturing respectively.Human lens capsule epithelium specimens were obtained at cataract surgery. Figs. (1A, B) show the same specimen, derived from an 85-year-old woman, after 1 week (A) and 2 weeks (B) respectively. The size of the capsule before outgrowth of epithelial cells was 5 mm. Lens epithelium cells were stained with the non-toxic fluorogenic compound carboxy-fluorescein diacetate succinimidyl ester (CFDA SE) to facilitate monitoring of cell growth. Original magnification x20 is shown.
Mentions: After one week, cultured cells were incubated with carboxy-fluorescein diacetate succinimidyl ester (CFDA SE) for 15 minutes at room temperature. CFDA SE (Vybrant CFDA SE, Invitrogen, Eugene, OR, USA) is a fluorogenic compound that, upon entering metabolically active cells, makes them fluorescent (excitation 492 nm, emission 517 nm). Being non-toxic to the cells, CFDA SE does not affect further growth of the cells, enabling continuous monitoring of proliferation. One week after the first staining procedure, however, the fluorescence had faded and cells were again incubated with CFDA SE. Prior to staining, a stock solution of 10 mM CFDA SE was prepared in dimethyl sulfoxide (DMSO). This stock solution was then further diluted to 10 µM in preheated phosphate buffered saline (PBS). After 15 minutes the CFDA SE solution was replaced by freshly prepared cell culture medium with 5% FCS and lens capsule epithelium specimens were viewed and photographed in a confocal microscope (Nikon Eclipse TE300 C1) using a Nikon D-Eclipse C1 camera and EZ-C1 3.30 Gold as imaging software. Fig. (1) shows an example of a capsulorhexis specimen after staining with CFDA SE after 1 and 2 weeks of culture respectively.

Bottom Line: Lower age and female sex showed border-line significant associations with a higher rate of cell proliferation.The presence of pseudoexfoliation in vivo did not significantly affect cell growth in culture postoperatively.The lack of strong correlations between in vitro growth and known risk factors for PCO in the donors suggest that other factors than the proliferative capacity of the cells per se are important for PCO formation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience and Physiology, Section of Clinical Neuroscience and Rehabilitation/Ophthalmology.

ABSTRACT

Aim: Inter-individual differences in intrinsic proliferative capacity of lens epithelial cells may have importance for the risk of developing posterior capsule opacification (PCO) after cataract surgery. The purpose of the present study was to determine growth of human lens epithelial cells (HLEC) in culture and investigate possible associations with clinical characteristics of the donors, such as age, sex, pseudoexfoliation, uveitis and diabetes.

Methods: Pieces of lens capsule and adhering lens epithelial cells were obtained through capsulorhexis at cataract surgery. Specimens were cultured in a humidified CO2-incubator using standard culture medium and 5% fetal calf serum for two weeks after which cultured cells were stained with carboxy-fluorescein diacetate succinimidyl ester. Image processing software was used to determine the area of the confluent epithelial cell layer in relation to the size of the original capsule specimen.

Results: The increase in area of confluent HLEC showed a negative correlation with diabetes at the first week after surgery. Lower age and female sex showed border-line significant associations with a higher rate of cell proliferation. The presence of pseudoexfoliation in vivo did not significantly affect cell growth in culture postoperatively. Nor did installation of xylocain in the anterior chamber during surgery.

Conclusion: Diabetes is associated with lower rate of proliferation of lens epithelial cells in culture. The lack of strong correlations between in vitro growth and known risk factors for PCO in the donors suggest that other factors than the proliferative capacity of the cells per se are important for PCO formation.

No MeSH data available.


Related in: MedlinePlus