Effect of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of in vitro cultivated limbal epithelial cell sheets

Abstract

PURPOSE:The aim of this study is to evaluate the effects of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of rabbit limbal stem cells cultivated in vitro, and to find out the proper timing of air-lifting before transplantation as limbal epithelial cell sheets for the treatment of limbal insufficiency.MATERIALS AND METHODS:Limbal epithelial cells were isolated from the limbus of New Zealand white rabbits and cultivated in vitro. After the cells became confluent, different durations of air-lifting (0, 1, 2, 4, and 7 days) were performed. At the end of cultivation, immunohistochemistry on cryosections was performed and observed by fluorescein microscopy and in vitro confocal microscopy for cytokeratins (K3, K10, K12, K13, and K14), junctional and structural proteins (ZO-1, p120, and actin) and stem cell markers (ABCG2 and P63). Scanning electron microscopy was used for observing the microstructure of superficial cells. Transepithelial electrical resistance (TEER) was used to measure the transepithelial permeability.RESULTS:The expression of K3, K10, K12, K13, K14, and ABCG2 showed no differences in pattern and location among different groups of airlifting. A time-dependent increase in corneal epithelial thickness was found after air-lifting. In vitro confocal microscopy demonstrated that K3, p120, and ZO-1 were expressed on the apical cell layer, whereas P63 and ABCG2 were expressed more on the basal epithelial layer. Scanning electron microscopy of the superficial layer demonstrated that airlifting induced time-dependent increase in the size of surface epithelial cells and triggered cellular differentiation. TEER results demonstrated a time-dependent increase of transepithelial electric resistance.CONCLUSIONS:During limbal epithelial cell expansion in vitro, air-lifting can increase cellular stratification, enlarge surface cells, trigger cellular differentiation, and increase the transepithelial barrier. However, the expression of cellular junctional, stem cell and cytokeratin markers seems to have no obvious differences in pattern and localization.

Notch Inhibition Prevents Differentiation of Human Limbal Stem/Progenitor Cells in vitro

“…Air-lifting has proven to reduce intercellular spaces in the superficial cells and promotes the formation of the barrier function 51 . Moveover, air-lifting has been shown to increases stratification, enlarge surface cells, triggers cellular differentiation and increases transepithelial electrical resistance, while maintaining the stem/progenitor cell properties at the basal layer 52 . By contrast, air-lifting was proposed to induce epithelial to mesenchymal transition (EMT) of LSCs and instrastromal invasion 53 when cultured with epithelial-stromal tissue recombinants.…”

Submicron spatial resolution optical coherence tomography for visualising the 3D structures of cells cultivated in complex culture systems

“…Furthermore, the ImageJ plugin 3D viewer was applied to reconstruct volumetric images. www.nature.com/scientificreports/ previous study 1 . In brief, limbal stem cells were extracted from the limbal area of adult New Zealand rabbits after euthanasia.…”

“…The cell culture model is increasingly valued in medical research, and various complex culture systems and three-dimensional (3D) culture systems are under development 1 , 2 . Cell culture systems can be used in lieu of experimental animals and provide a large number of consistent and reliable results for biological assays.…”

“…First, we demonstrated that cultivated N2A neuron cells and their thin neuron processes could be easily observed using OCT. Second, we observed multilayer cultivated limbal epithelial sheets (mCLESs) prepared for transplantation to treat limbal insufficiency. In this cell culture system, airlifting, a well-documented method for promoting epithelium stratification 1 , was performed following the confluence of the cultivated single layer cell sheet [14][15][16][17][18][19] . Multilayered cell products prevented the damage of fragile cultivated cells from external stimuli after transplantation to the ocular surface of patients.…”

Differentiation Induction of Human Stem Cells for Corneal Epithelial Regeneration

“…Limbal stem cells, the stem cells residing in the limbus, generate new corneal epithelial cells to replace the shed old cells or damaged cells to maintain the corneal epithelial population. Severe damage, severe microbial infections, or some diseases such as Stevens–Johnson syndrome and ocular cicatricial pemphigoid [ 2 , 3 ] may cause the depletion of limbal stem cells. Such a condition is called limbal stem cell deficiency (LSCD).…”