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mafrici marco, Paolo Trabucco, Stefano Valente, Leopoldo Spadea, Enzo M Vingolo; Experimental surgical technique in isolation of limbal stem cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):521.
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The purpose of this study is to describe a standardized method of limbal tissue sampling. This is important to maximize the stem cells content, permitting to researchers to work with these samples. The epithelium of the ocular surface, corneal and conjunctival epithelia, is constantly renewed through processes of proliferation and differentiation, and these mechanisms are active both in basal conditions and after injury. The proliferative potential is a matter exclusively of stem cells. The corneal limbus, as demonstrated by several studies in the literature, is an area particularly rich in stem cells P63 positive, especially in upper sector.
113 samples were taken from the limbus of 113 patients operated for retinal detachment, glaucoma and strabismus. Samples were taken before surgical operation. Peribulbar local anesthesia with ropivacaine(5ml at 2%) was performed before taking the sample. Disinfection of operating field was performed with iodopovidone (5%) for 10 seconds. The sample was collected in the upper limbus, 12mm along the corneal margin and 3mm towards the conjunctival fornix, from 9 o’clock to 3 o’clock, in upper sector. The samples were transported in sterile tubes(temperature: 20°-25°).
We have the limbal cells extract samples from 113 limbal getting an average of 310,000 cells, with a maximum of 1.3 X106 cells. The vitality of cells taken amounted to 98%. We have found that this technique allows to achieve results in terms of the number of cells collected and limbal cell vitality
In this study we described a standardized method to collect a limbal sample. The method described permits to have limbal samples that contains a good cellular number and a good cellular vitality, therefore, permitting also to form several cellular cultures. Limbal stem cells, in next future, will be used in regenerative medicine, especially, in corneal wound healing. A good sample is the starting point to achieve a successful cellular experiment. The authors hope that this method is a contribution in corneal wound healing research.
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