Growth Of Limbal Epithelial Cells Research Articles

Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro - A Stepwise Linear Regression Algorithm.

To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm. Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO2 atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm. The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007). The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro.

CREATING CORNEAL STEM CELLS WITH A THEROREVERSIBLE GELATION POLYMER

Introduction: Recent technological advancements in single-cell RNA sequencing and single-cell quantitative real-time PCR have enabled researchers to conduct single-cell analyses of limbal epithelial cells. Aim of the study: the main aim of the study is to Cultivating corneal stem cells with a thermoreversible gelation polymer Material and method: Each human limbal biopsy were placed in 1 mL of TC medium containing 3% FCS, antibiotics (penicillin, gentamicin, and amphotericin B), and DMEM, and then transported to a cell biology lab. Conclusion: The thermoreversible gelation polymer facilitates the growth of limbal epithelial cells. The limbal characteristic could still be seen in the grown cells

Ex Vivo Expansion of Human Limbal Epithelial Cells Using Human Placenta-Derived and Umbilical Cord-Derived Mesenchymal Stem Cells.

Ex vivo culture of human limbal epithelial cells (LECs) is used to treat limbal stem cell (LSC) deficiency, a vision loss condition, and suitable culture systems using feeder cells or serum without animal elements have been developed. This study evaluated the use of human umbilical cord or placenta mesenchymal stem cells (C-MSCs or P-MSCs, resp.) as feeder cells in an animal/serum-free coculture system with human LECs. C-/P-MSCs stimulated LEC colony formation of the stem cell markers (p63, ABCG2) and secreted known LEC clonal growth factors (keratinocyte growth factor, β-nerve growth factor). Transforming growth factor-β-induced protein (TGFBIp), an extracellular matrix (ECM) protein, was produced by C-/P-MSCs and resulted in an increase in p63+ ABCG2+ LEC colonies. TGFBIp-activated integrin signaling molecules (FAK, Src, and ERK) were expressed in LECs, and TGFBIp-induced LEC proliferation was effectively blocked by a FAK inhibitor. In conclusion, C-/P-MSCs enhanced LEC culture by increasing growth of the LSC population by secreting growth factors and the ECM protein TGFBIp, which is suggested to be a novel factor for promoting the growth of LECs in culture. C-/P-MSCs may be useful for the generation of animal-free culture systems for the treatment of LSC deficiency.

Effect of Cryopreserved Amniotic Membrane Orientation on the Expression of Limbal Mesenchymal and Epithelial Stem Cell Markers in Prolonged Limbal Explant Cultures.

PurposeTo evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC).MethodsLimbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67.ResultsMorphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM.ConclusionLimbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67.

Decellularized pig corneal stroma seeded with limbal cells

Event Abstract Back to Event Decellularized pig corneal stroma seeded with limbal cells Wendolyne Lara Daz1, Beatriz Hernandez Tellez1, Judith Alvarez-Perez1, Katia Jarquín-Yanez1, Gabriela Piñón-Zarate1, Sandra Acevedo Nava1, Miguel A. Herrera-Enríquez1 and Andrés Castell-Rodriguez1 1 UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO, Cell and tissue biology, Medicine School, Mexico Introduction: The cornea is a highly specialized avascular organ in the anterior surface of the eye. It provides two thirds of the refractive capacity thereof while shielding structures acting as a barrier to infectious agents and mechanical damage. The cornea consists of five layers: anterior epithelium, Bowman's membrane, stroma, Descemet's membrane and posterior epithelium (or corneal endothelium). In the transition zone between the cornea and sclera (limbus) resides a subpopulation of corneal stem cells has been observed, exhibit a high proliferative potential and have the ability to differentiate into corneal epithelial cells, called limbal epithelial cells. Various diseases can damage the cornea leading to corneal opacity and once this happens transplantation is the predominant choice to correct visual impairment. Tissue engineering allows the creation of tissue equivalents with cultured cells in an extracellular matrix and growth factors. Corneal cells have been cultivated in different types of three-dimensional matrices as fibrin, collagen and synthetic polymers, however they present several disadvantages as rapid degradation of the scaffold and inflammation. While devitalized stroma not only possess adequate formed, but show binding sites integrins located in the limbal epithelial cells, which are an alternative to the development of a corneal equivalent it may be used as a potential source of xenografts viable cornea for transplantation. Objective: The aime of this study was to induce the growth of limbal epithelial cells in a porcine decellularized corneal stroma in vitro. Materials and Methods: Anterior and posterior corneal limbal epithelial cells were isolated and expanded, corneal stromas were obtained and devitalized using Triton X100 solution. The latter was histologically analyzed to check absence of cells and α-gal epitope. Epithelial cells were seeded and cultured in the anterior portion and the posterior endothelial cells in decellularized portion stroma. Further histological slices were obtained and processed to detect the presence of cytokeratin immunohistochemistry. Results: Histological analysis of devitalized the absence of stroma cells were found within the stroma and α-gal. Limbal epithelial cells proliferated on the anterior surface of the stroma decellularized and formed a confluent layer of cells two or three layers thick, whereas endothelial cells and later joined epithelium expanded on the stromal surface to confluence and form a monolayer. Immunohistochemical analysis revealed the presence of cytokeratins in the epithelium. Conclusions: limbal epithelial cells seeded on the decellularized porcine stroma were able to proliferate and differentiate to give rise to the generation of an earlier and a later epithelium. It is important to conduct more studies in this type of constructs for the future may be used in people with corneal opacities. Acknowledgements: DGAPA PAPIIT IN218315. DGAPA- PAPIIT IN218315

Photo-cross-linking of amniotic membranes for limbal epithelial cell cultivation

In the present study, we developed photo-cross-linked amniotic membrane (AM) as a limbal stem cell niche. After ultraviolet (UV) irradiation for varying time periods, the biological tissues were studied by determinations of cross-linking structure, degradability, and nutrient permeation ability. Our results showed that the number of cross-links per unit mass of AM significantly increased with increasing illumination time from 5 to 50min. However, the cross-link formation was inhibited by longer irradiation time (i.e., 150min), probably due to the scission of tissue collagen chains through irradiation. The biological stability and matrix permeability of photo-cross-linked AM materials strongly depended on their cross-linking densities affected by the UV irradiation. In vitro biocompatibility studies including cell viability and pro-inflammatory gene expression analyses demonstrated that, irrespective of the irradiation time employed, the physically cross-linked biological tissues exhibited negligible cytotoxicity and similar interleukin-6 (IL-6) mRNA levels. The data clearly indicate that these AM matrices do not cause potential harm to the corneal epithelial cells. After the growth of limbal epithelial cells (LECs) on AM substrates, Western blot analyses were conducted to examine the expression of ABCG2. It was found that the ability of UV-irradiated AM to maintain the undifferentiated precursor cell phenotype was significantly enhanced with increasing extent of photo-cross-linking. In summary, the UV irradiation time may have a profound influence on the fabrication of photo-cross-linked AM matrices for LEC cultivation.

Effect of Matrix Nanostructure on the Functionality of Carbodiimide Cross-Linked Amniotic Membranes as Limbal Epithelial Cell Scaffolds

Carbodiimide cross-linked amniotic membrane (AM) can potentially serve as an artificial corneal epithelial stem cell niche in ocular surface wound healing. For the first time, this study was performed to investigate the relationship between nanostructure and functionality of carbodiimide cross-linked AM tissues as limbal epithelial cell (LEC) scaffold biomaterials. The triple-helical molecular conformation of AM collagen was checked after chemical treatment for varying cross-linking durations (1-4 h). Our data indicated that the unraveling of the helical structure into a more random globular state is accompanied by an increase in the cross-linking index of AM samples. The cross-linker-mediated alterations in tissue ultrastructure and substrate nanotopography of these proteinaceous matrices were confirmed by transmission electron and atomic force microscopy studies. With increasing treatment time, the chemically cross-linked AM possessed larger nanofiber diameter and exhibited rougher texture. Marked increases in the water content, light transmittance, and resistance to enzymatic degradation were found, probably due to collagen fibril aggregation in biological tissues. All the test AM materials were not toxic to the human corneal epithelial cell cultures and retained anti-inflammatory activity, indicating the tolerability and safety of carbodiimide (i.e., a zero-length cross-linker). In addition, the enhanced LEC growth and increased p63 and ABCG2 gene expressions were significantly noted on the AM samples with greater cross-linking degree. In summary, the findings reported in this paper suggest that a specific limbal epithelial stem cell-biomaterial interaction may occur in response to biophysical cue such as nanostructure of carbodiimide cross-linked AM matrix.

Ex VivoConstruction of an Artificial Ocular Surface by Combination of Corneal Limbal Epithelial Cells and a Compressed Collagen Scaffold Containing Keratocytes

We have investigated the use of a laminin-coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane (AM). Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibers within the compressed gel were dense, homogeneous, and similar in structure to those within denuded AM. Limbal epithelial cells were successfully expanded upon the compressed collagen, resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology, and cell stratification. Similar protein expression profiles for cytokeratin 3 (CK3) and CK14 and no significant difference in CK12 mRNA expression levels by real-time polymerase chain reaction were also observed. This study provides the first line of evidence that a laminin-coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification, and differentiation to a degree that is comparable to the leading conventional scaffold, denuded AM.