Limbal Cultures Research Articles

SOX2 Is a Univocal Marker for Human Oral Mucosa Epithelium Useful in Post-COMET Patient Characterization.

Total bilateral Limbal Stem Cells Deficiency is a pathologic condition of the ocular surface due to loss or impairment of corneal stem cell function, altering homeostasis of the corneal epithelium. Cultivated Oral Mucosa Epithelial Transplantation (COMET) is the only autologous treatment for this pathology. During the follow-up, a proper characterization of the transplanted oral mucosa on the ocular surface supports understanding the regenerative process. The previously proposed markers for oral mucosa identification (e.g., keratins 3 and 13) are co-expressed by corneal and conjunctival epithelia. Here, we propose a new specific marker to distinguish human oral mucosa from the epithelia of the ocular surface. We compared the transcriptome of holoclones (stem cells) from the human oral mucosa, limbal and conjunctival cultures by microarray assay. High expression of SOX2 identified the oral mucosa vs. cornea and conjunctiva, while PAX6 was highly expressed in corneal and conjunctival epithelia. The transcripts were validated by qPCR, and immunological methods identified the related proteins. Finally, the proposed markers were used to analyze a 10-year follow-up aniridic patient treated by COMET. These findings will support the follow-up analysis of COMET treated patients and help to shed light on the mechanism of corneal repair and regeneration.

Comparison of culture media indicates a role for autologous serum in enhancing phenotypic preservation of rabbit limbal stem cells in explant culture.

In this study, we aimed to compare the effects of six different cell culture media and autologous serum (AS) on the phenotypic characteristics of rabbit limbal epithelial stem cells (LESC) cultivated on porous polyethylene terephthalate (PET) membranes. Limbal explants from rabbit corneas were grown on PET membrane inserts in five different media: DMEM-F12 with fetal bovine serum (FBS) (DMEM-F12-FBS), with pluripotin (DMEM-F12-pluripotin) and with autologous serum (DMEM-F12-AS), Epilife, Keratinocyte Serum Free Medium (KSFM) and Defined-Keratinocyte Serum Free Medium. The effects of different media were evaluated by total cell yield from explants, measuring the expression of proteins by immunofluorescence and gene expression by Real Time PCR. In all five media tested, most of the limbal epithelial cells (LEC) which proliferated from explants were positive for cytokeratin (CK) 14 (85-90%), indicating that all five media support the growth of LESC from explants. The expression of differentiation markers; CK 3 and 12 was highest in DMEM-F12-FBS (56%), was lower in Epilife and KSFM (26 and 19%, respectively), with the lowest values (13%) obtained in DMEM-F12-AS. Gene expression of limbal cultures on PET membrane inserts was compared to fresh limbal tissue. In DMEM-F12-FBS, DMEM-F12-pluripotin, and DMEM-F12-AS, expression of potential LESC markers CXCR4 and polycomb complex protein BMI-1 were similar to limbal tissue. DMEM-F12 with 10% AS maintained a higher percentage of potential stem cell marker genes and lower expression of genes involved in differentiation compared to Epilife or KSFM. Our study shows that rabbit LEC can be cultivated on PET inserts using DMEM-F12 with autologous serum without a requirement for amniotic membrane or feeder cells.

Cultivated limbal epithelial transplantation.

To provide an updated literature review on the status of cultivated limbal (corneal) epithelial transplantation. Cultivated limbal stem-cell transplantation recently received regulatory approval. We provide a comprehensive overview of recent developments in the field. The current article reviews and highlights recent developments in the field of cultivated limbal stem-cell transplantation as retrieved from a literature search for the last year. The implications of clinical/research findings in terms of transplanted cell source and cultivation methods in limbal stem-cell transplantation are reviewed.

Human limbal neurospheres prevent photoreceptor cell death in a rat model of retinal degeneration.

The culture of retinal progenitors from an accessible adult stem cell source such as the limbus could provide a useful autologous source of retinal cell therapies. The human corneoscleral limbus contains multipotent stem cells that can be cultured as floating neurospheres. Previous work in rodents has demonstrated neuronal and photoreceptor differentiation from limbal neurosphere cultures. Here, this study has examined undifferentiated cultured adult human limbal neurospheres as donor cells for retinal cell therapies by transplantation into a rat model of retinal degeneration. Gene expression in limbal neurospheres was examined by immunostaining and western blot. Human limbal neurospheres were transplanted into the subretinal space of Royal College of Surgeon's rats. Rats were monitored by optical coherence tomography for 6weeks then processed for retinal histology. Human limbal neurospheres expressed the neural lineage markers, Nestin, sex determining region box-2 and N-cadherin, and the retinal transcription factors microphthalmia-associated transcription factor, sex determining region box-2 and orthodentical homeobox-2. Human limbal neurospheres could be cultured to express NeuN, neurofilament and rhodopsin. Rats receiving saline or no injection underwent complete degeneration of the retinal outer nuclear layer after 3weeks. In contrast, rats injected with human limbal neurospheres or retinal pigment epithelial cells maintained the outer nuclear layer for up to 6weeks. Gene expression in transplanted limbal neurospheres was inconsistent with the production of mature retinal pigment epithelial or photoreceptor cells. Human limbal neurospheres represent an accessible source of autologous donor cells for the treatment of retinal diseases.

Limbal stem cell and oral mucosal epithelial transplantation from ex vivo cultivation in LSCD-induced rabbits: histology and immunologic study of the transplant epithelial sheet.

To evaluate the results of cultivated limbal epithelial and oral mucosal epithelial transplantation (CLET and COMET) in limbal stem cell deficiency (LSCD)-induced rabbit model. Six New Zealand white rabbits were divided into two groups of three rabbits each. Limbal tissue was harvested from the first group, and oral mucosal biopsy was obtained from the second group. The tissues were cultured using an explant technique with amniotic membrane as a substrate and co-culture with the 3T3 fibroblast and air-lifting method. The right eye of each rabbit was induced to have LSCD using alkali burns. After three weeks, the LSCD-induced rabbit eyes were transplanted with the cultivated limbal and oral mucosal epithelial sheet in the first and second group, respectively. The transplanted eye was evaluated weekly post-operation. After 2 months, all transplanted eyes were enucleated and the epithelial morphology and phenotype of ocular surfaces were studied and compared with normal corneal and oral mucosal tissue. At 2-month post-transplantation, the eyes of four animals recovered with corneal transparency, one partially recovered, and one failed. The histology of the majority of transplanted eyes was stratified layers of corneal epithelia similar to normal rabbit cornea with some different findings such as goblet cells in the limbal region. Corneal epithelial thickening and stromal vascularization in two animals were observed. Phenotypic characterization of transplanted eyes showed a similar pattern of marker expression with the absence of p63 expression in the limbal or corneal epithelium in the COMET group. The histology and phenotype of transplanted eyes after CLET and COMET were most likely to have similar characteristics as a normal healthy rabbit eye even though the COMET eyes have some inferior characteristics to the CLET eyes.

Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions

ABSTRACTPurpose: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers.Materials and methods: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence.Results: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished.Conclusion: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.

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.

Comparison of functional limbal epithelial stem cell isolation methods

The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either of the isolation techniques from the donor limbal tissue, then the cell suspension is probably the best option as long as the cells are expanded following our culture conditions.

Spatial Distribution of Niche and Stem Cells in Ex Vivo Human Limbal Cultures

Stem cells at the limbus mediate corneal epithelial regeneration and regulate normal tissue homeostasis. Ex vivo cultured limbal epithelial transplantations are being widely practiced in the treatment of limbal stem cell deficiency. In this report, we examined whether the limbal niche cells that nurture and regulate epithelial stem cells coexist in ex vivo limbal cultures. We also compared the inherent differences between explant and suspension culture systems in terms of spatial distribution of niche cells and their effect on epithelial stem cell proliferation, migration, and differentiation in vitro. We report that the stem cell content of both culture systems was similar, explaining the comparable clinical outcomes reported using these two methods. We also showed that the niche cells get expanded in culture and the nestin-positive cells migrate at the leading edges to direct epithelial cell migration in suspension cultures, whereas they are limited to the intact niche in explant cultures. We provide evidence that C/EBPδ-positive, p15-positive, and quiescent, label-retaining, early activated stem cells migrate at the leading edges to regulate epithelial cell proliferation in explant cultures, and this position effect is lost in early suspension cultures. However, in confluent suspension cultures, the stem cells and niche cells interact with each another, migrate in spiraling patterns, and self-organize to form three-dimensional niche-like compartments resembling the limbal crypts and thereby reestablish the position effect. These 3D-sphere clusters are enriched with nestin-, vimentin-, S100-, and p27-positive niche cells and p15-, p21-, p63α-, C/EBPδ-, ABCG2-, and Pax6-positive quiescent epithelial stem cells.

Cultivation and Phenotypic Characterization of Rabbit Epithelial Cells Expanded Ex Vivo from Fresh and Cryopreserved Limbal and Oral Mucosal Explants

Purpose: To compare the morphology of cultured rabbit epithelial sheets and the expression of stem cells with differentiated cell markers of cultivated epithelial cells from fresh and cryopreserved limbal and oral mucosal biopsies.Materials and methods: Six New Zealand white rabbits were divided into two groups of three, from which limbal and oral mucosal biopsies were taken. Harvested tissues from each rabbit were brought to immediate cultivation, while another set of tissues was cryopreserved. Cultivation was performed by the explant culture technique using human amniotic membrane as a culture substrate, co-culturing with 3T3 fibroblasts and using the air-lifting method. Cells were cultured for three weeks; then cultured epithelial sheets were stained with hematoxylin–eosin and examined for expression patterns of p63, keratin 3 (K3) and connexin 43 (Cx43). Cryopreservation was carried out using the vitrification method. Tissues were preserved in liquid nitrogen using 25% dimethyl sulfoxide combined with 25% propylene glycol in Dulbecco’s Modified Eagle’s Medium containing 20% fetal bovine serum. After two months, the tissues were warmed, cultured and stained using the same processes as for fresh tissue cultures.Results: Cultivation of fresh limbal and fresh oral mucosal tissues showed epithelial stratification, with two to five cell layers. Immunohistochemical staining showed p63-positive cells in basal and intermediate cell layers. K3 staining was observed in cells in the suprabasal layer, while expression of Cx43 was scattered throughout all layers of the epithelia. All culture sheets expressed p63, K3 and Cx43 with the exception of one sheet from the oral mucosal culture that was p63-negative. Cultured epithelial sheets from cryopreserved tissues showed results similar to those from fresh tissue culture.Conclusions: This study found that cells in cultivated fresh limbal and oral mucosal tissues had similar morphology to cells in cultivated cryopreserved limbal and oral mucosal tissues, both containing a heterogeneous population of cells including stem cells and differentiated cells.

Evaluation of ABCG2 and p63 expression in canine cornea and cultivated corneal epithelial cells

To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration. Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR. The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages. ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively.

Characterisation of Human Limbal Side Population Cells Isolated Using an Optimised Protocol From an Immortalised Epithelial Cell Line and Primary Limbal Cultures

The challenges in limbal stem cell biology largely remain in the process of identification, isolation and expansion of these adult corneal epithelial stem cells of the eye. Due to the absence of specific limbal stem cell markers, identification and isolation of putative limbal stem cells is a complicated task. The side population assay is an isolation method that utilises the ability of stem cells to efflux the DNA-binding dye Hoechst 33342 (or other vital dyes) combined with dual wavelength flow cytometry and is a valuable strategy to enrich for limbal stem cells. This assay has been used to successfully identify stem/ progenitor cell populations in a variety of tissues and cell lines. Here we optimise this assay to identify SP cell populations in both primary human limbal epithelial cultures and in an established human corneal epithelial cell line. The limbal SP fraction showed higher expression of ATP-binding cassette sub-family G member 2 (ABCG2), ΔNp63--a common limbal stem cell marker and the stem cell marker Sox2 compared to non-SP cells (NSP).

Biological Parameters Determining the Clinical Outcome of Autologous Cultures of Limbal Stem Cells

Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigated the biological parameters instrumental for their clinical success. We report a long-term multicenter prospective study on 152 patients carrying corneal destruction due to severe ocular burns, treated with autologous limbal cells cultured on fibrin and clinical-grade 3T3-J2 feeder cells. Clinical results were statistically evaluated both by parametric and nonparametric methods. Clinical outcomes were scored as full success, partial success and failure in 66.05, 19.14 and 14.81% of eyes, respectively. The total number of clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results. Instead, the clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones expressing high levels of the p63 transcription factor. No adverse effects related to the feeder layer have been observed and the regenerated epithelium was completely devoid of any 3T3-J2 contamination. Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the human cornea. We emphasize the importance of a discipline for defining the suitability and the quality of cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.

Simplifying corneal surface regeneration using a biodegradable synthetic membrane and limbal tissue explants

Currently, damage to the ocular surface can be repaired by transferring laboratory cultured limbal epithelial cells (LECs) to the cornea using donor human amniotic membrane as the cell carrier. We describe the development of a synthetic biodegradable membrane of Poly d,l-lactide-co-glycolide (PLGA) with a 50:50 ratio of lactide and glycolide for the delivery of both isolated LECs and of cells grown out from limbal tissue explants. Both isolated LECs and limbal explants produced confluent limbal cultures within 2 weeks of culture on the membranes without the need for fibroblast feeder layers. Outgrowth of cells from explants was promoted by the inclusion of fibrin. Membranes with cells on them broke down predictably within 4–6 weeks in vitro and the breakdown was faster for a lower molecular weight (MW) (44 kg/mol) rather than a higher MW (153 kg/mol) PLGA. Membranes could be reproducibly produced, sterilised with gamma irradiation and stored dry at −20 °C for at least 12 months, and the ability to support cell outgrowth from explants was retained. We demonstrate transfer of cells (both isolated LECs and of cells grown out from limbal explants) from the membranes to an ex vivo rabbit cornea model. Characterisations of the cells by immunohistochemistry showed both differentiated and stem cell populations. A synthetic membrane combined with limbal explants in theatre would avoid the need for tissue banked human amniotic membrane and also avoid the need for specialist laboratory facilities for LEC expansion making this more accessible to many more surgeons and patients.

Consecutive Expansion of Limbal Epithelial Stem Cells from a Single Limbal Biopsy

Purpose: Corneal epithelium is maintained by limbal epithelial stem cells (LESCs), the loss of which can be catastrophic for corneal transparency. Effective therapies include the transplantation of cultivated LESCs, requiring optimization of in vitro cultivation protocols. Unfortunately, optimization studies are hampered by the limited number of ocular tissue donors. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same 1-2 mm2 limbal explant (LE).Methods: LEs were plated and maintained until outgrowth surrounded each, being removed at this point. LPCs were allowed to reach confluence (LPC0). The same removed LE was plated again, following the same procedure, obtaining LPC1. This procedure was repeated as often as possible up to six times. LPCs from each passage were analysed by real time reverse transcription-polymerase chain reaction and immunofluorescence-microscopy.Results: LPCs from LPC0 to LPC2 presented a heterogeneous cell population, with cells positive for LESC markers K14, K15, ABCG2 and p63, differentiated corneal epithelial cell-specific markers K3 and K12, and for the fibroblast marker S100A4. These cells had an epithelial-like morphology. In LPC3-LPC4, elongated cell morphology appeared, and the presence of LESC markers decreased, while the presence of differentiated corneal epithelial-cell and fibroblast markers increased.Conclusion: One LE can be successfully cultivated up to three consecutive times while maintaining the LESC phenotype in the LPC cells. This protocol provides several homologous LPCs for basic research. Additionally, by using a cell-carrier, the resulting LPCs could serve reservoirs for potential autologous expanded LESC transplantations and/or for making correlations between laboratory and clinical outcomes.

Expression of human limbal epithelial stem cells markers and mRNA in long term repeated limbal cultures

Expression of human limbal epithelial stem cells markers and mRNA in long term repeated limbal cultures

Preparation of Corneal Donor Eyes Comparing 1% Versus 5% Povidone–Iodine

To compare the effect of 1% versus 5% polyvinylpyrrolidone-iodine (PVP-I) chemical preparation (prep) of the eye on the recovery of organisms from donor globes before in situ recovery of donor corneal tissue. One hundred consecutive pairs of donor corneas (200 eyes) were randomized to receive either 1% or 5% PVP-I drops applied to the conjunctival cul-de-sac, which was left in place for 2 minutes. Limbal cultures were obtained before and after prepping of the eye. Twenty-five different species of organisms were recovered. Native flora of the eye included coagulase-negative staphylococci (62%), Corynebacterium species (27%), streptococcal species (9.5%), gram-negative bacilli (14.5%), Staphylococcus aureus (5%), anaerobes (10%), and yeast (2%). After PVP-I instillation of the donor eye, 74 isolates were recovered from the 1% P-I group and 76 isolates from the 5% PVP-I group. Cultures were sterile after PVP-I prep in 49 eyes and 47 eyes in the 1% PVP-I group and 5% PVP-I group, respectively. Microorganism colony forming units were similar among post-prep cultures from both PVP-I groups. The effect of the PVP-I prep on the number of negative cultures and on the reduction in the number of isolates was highly significant for both the 1% PVP-I group and the 5% PVP-I group when compared with the limbal cultures taken before PVP-I instillation. This study found that 1% and 5% PVP-I solutions are equally effective for chemical prep of the donor eye. Because PVP-I is known to be toxic to the corneal endothelium and corneal fibroblasts, this study suggests that 1% PVP-I should be the preferred disinfectant for the recovery of corneal donors.

Vision from the right stem

Cultures of limbal cells are a safe and effective treatment for the destruction of the human cornea owing to chemical burns. The essential feature of the graft is the presence of an adequate number of stem cells, which can be determined by the expression of the p63 transcription factor. Here, we will discuss the general principles defining the rigorous criteria for graftable limbal cultures in light of their clinical performances. Such criteria might prove relevant to the future therapeutic use of any cultured cell type.

Techniques for Culture and Assessment of Limbal stem Cell Grafts

The therapeutic use of limbal cultures for the permanent regeneration of corneal epithelium in patients with limbal stem cell deficiency (LSC D) has been reported in many studies. A ccording to the guidelines for good manufacturing practice (GMP), strictly regulated procedures and stringent quality control tests are now required to manipulate stem cells as “medicinal products” and make engraftment safer and eventually more successful. This paper describes techniques for optimal preparation of limbal stem cell grafts, including 1) a reliable impression cytology assay for the grading of LSCD, 2) culture methods that maintain high percentages of limbal stem cells, 3) the use of specific markers for the detection of corneal, conjunctival, and limbal stem cells, namely keratin 12, mucin 1, and ΔNp63α, and 4) assays to assess the presence of contaminants, such as murine fibroblasts, endotoxins, mycoplasmae, and viral particles, in the cultured graft. The use of some of these assays allowed us to obtain a regenerated normal corneal epithelium in approximately 80% of 166 LSCD patients who received transplants from 2004 to 2008.

Optimization of culture conditions for an efficient xeno‐feeder free limbal cell culture system towards ocular surface regeneration

Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever-increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno-diseases to human system. This study was aimed to establish an efficient xeno-feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using "interactive measurements" of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno-feeder free epithelial equivalents towards ocular surface reconstruction.