Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue.

PURPOSE: To examine levels of oxidative DNA base damage and expression of selected genes and proteins related to DNA damage repair in human limbal epithelium engineered ex vivo. METHODS: Cells were expanded from limbal tissue on cell culture-treated inserts in medium containing fetal bovine serum, recombinant growth factors, hormones and cholera toxin (COM) and in medium with human serum as the single growth-promoting additive (HS). Cells were analysed after two, three and four weeks in culture for DNA strand breaks and oxidized purine bases (Comet assay using the enzyme formamidopyrimidine DNA glycosylase, Fpg) and for expression of DNA repair enzymes APE1, OGG1 and Polß by in situ hybridization (ISH) and by immunohistochemistry (IHC). RESULTS: Levels of strand breaks were substantial while levels of net Fpg-sensitive sites (8-oxoguanine and ring-opened FaPy bases) were relatively low in cells engineered in COM and in HS. Both types of medium were found to support expression of base excision repair (BER) enzymes APE1, OGG1 and Polß at the gene level. At the protein level, expression of APE1 and OGG1 was noticeable in both conditions while expression of Polß was low. CONCLUSION: Our findings indicate low levels of oxidative stress and/or efficient DNA purine base damage repair in human limbal epithelium engineered in a medium with human serum as the single growth-promoting additive as well as in traditional medium with xenobiotics.

The comet assay applied to cells of the eye.

The human eye is relatively unexplored as a source of cells for investigating DNA damage. There have been some clinical studies, using cells from surgically removed tissues, and altered DNA bases as well as strand breaks have been measured using the comet assay. Tissues examined include corneal epithelium and endothelium, lens capsule, iris and retinal pigment epithelium. For the purpose of biomonitoring for exposure to potential mutagens in the environment, the eye-relatively unprotected as it is compared with the skin-would be a valuable object for study; non-invasive techniques exist to collect lachrymal duct cells from tears, or cells from the ocular surface by impression cytology, and these methods should be further developed and validated.

Proliferative Cells Isolated from the Adult Human Peripheral Retina only Transiently Upregulate Key Retinal Markers upon Induced Differentiation.

Purpose/Aim: The adult human retina has limited regenerative potential, and severe injury will result in permanent damage. Lower vertebrates handle retinal injury by activating neural stem cells (NSCs) in the ciliary marginal zone (CMZ). Müller glia-like cells expressing markers of NSCs are also present in the peripheral retina (PR) of the adult human eye, leading to the hypothesis that a CMZ-like zone might exists also in humans. In order to shed further light on this hypothesis we investigated the in vitro differentiation potential of proliferative cells isolated from the adult human PR towards a retinal phenotype. MATERIALS AND METHODS: Proliferative cells were isolated from the peripheral retina of human eyes (n = 6) within 24 to 48 hours post mortem and further expanded for 2 or 3 passages before being differentiated for 1-3 weeks. Gene expression was analyzed by microarray and qRT-PCR analysis, while protein expression was identified by immunocytochemistry. RESULTS: A high density of cells co-staining with markers for progenitor cells and Müller glia was found in situ in the PR. Cells isolated from this region and cultured adherently showed fibrillary processes and were positive for the immature marker Nestin and the glial marker GFAP, while a few co-expressed PAX6. After 7 days of differentiation, there was a transient upregulation of early and mature photoreceptor markers, including NRL, CRX, RHO and RCVRN, as well as the Müller cell and retinal pigmented epithelium (RPE) marker CRALBP, and the early RPE marker MITF. However, the expression of all these markers dropped from Day 14 and onwards. CONCLUSIONS: Upon exposure of proliferating cells from the adult human PR to differentiating conditions in culture, there is a widespread change in morphology and gene expression, including the upregulation of key retinal markers. However, this upregulation is only transient and decreases after 14 days of differentiation.

The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).

Cell death pattern in lens epithelium of cataract patients.

PURPOSE: Apoptosis, a type of programmed cell death, is observed in various types of cataract and in cultured lens epithelium subjected to oxidative damage. We have recently described oxidative DNA base damage in epithelium in age-related cataract and cultured cells, and we here aimed to examine such epithelium for markers for proliferation, initiation of apoptosis and morphological patterns of cell damage. METHODS: Samples (n = 75) were analysed by light microscopy/electron microscopy (LM/EM); immunohistochemistry (IHC) for PCNA and Ki67 (DNA synthesis/proliferation); TUNEL assay (DNA fragmentation/apoptosis); and protein/gene expression of Caspase-3 (apoptotic effector molecule) and BAX/Bcl2 (pro-/anti-apoptotic marker) in fresh/cultured epithelium by IHC and qRT-PCR. RESULTS: In fresh samples, the majority of cells were Ki67-/PCNA+. BAX/BCL-2-ratio was approximately 1, and Caspase-3 levels were low. TUNEL stained scattered nuclei/nuclear fragments (9/6302 cells). Main morphological signs of cell damage included rupture of cell membranes and hydration of cytoplasm and nuclei. Cultivation increased levels of BAX and Bcl2 by IHC and qRT-PCR (approximately 10-fold upregulation). Caspase-3 levels remained low by IHC with similar expression in fresh and cultured samples by qRT-PCR. CONCLUSION: Genomic stress and DNA repair may explain the contrasting expression of Ki67/PCNA in fresh epithelium. Despite low levels of Caspase-3 and similar expression of BAX/Bcl-2, a low incidence of apoptosis may be detected in epithelium in age-related corticonuclear cataract. Epithelium may be transferred to culture without an increase in expression of Caspase-3, one of the central mediators of apoptosis.

Surgical anatomy of the superior orbit on ultra-high-resolution MRI at 9.4 Tesla.

BACKGROUND: A good understanding of the anatomical details is required to ensure optimal results during surgery of the orbit. Several indications for orbital surgery require biopsy, resection, or reconstructive procedures. The intricate relationships between the orbital septum and adjacent structures of the upper orbit can cause difficulties in interpreting the surgical anatomy of this region. The purpose of this study was to acquire further insight into the anatomy of the superior part of the orbit, with special attention paid to the orbital septum. METHODS: An ex-vivo study was performed using magnetic resonance imaging (MRI) at 9.4 Tesla (isotropic resolution = 20 µm) on six human cadaver specimens to examine the superior-medial half of the orbit. To visualise the posterior layers of the upper orbit, a dissection of three of the orbits was performed prior to the MRI examination, and a flexible PVC sheet was introduced above the levator muscle. RESULTS: The technique enabled a visualisation of anatomically important landmarks of the anterior and posterior parts of the upper orbit at a resolution near histological levels; to the authors' knowledge, this visualisation has not been reported previously. A posterior continuation of the orbital septum, which forms a distinct anatomical structure, is revealed. CONCLUSIONS: The posterior aspect of the orbital septum separates the levator muscle and the orbital fat pad. Between these two structures, a surgical corridor is formed using MRI, enabling alternative access to the superior part of the orbit; this alternative access might be less invasive because the orbital septum remains undamaged.

Suspension surgery with autogenous fascia lata via a less invasive modification of the Crawford method on 85 patients with congenital severe eyelid ptosis.

BACKGROUND: Autogenous fascia lata is considered the gold standard for frontalis suspension surgery to correct severe congenital upper eyelid ptosis. METHODS: This study evaluated the efficacy of a less invasive modification of the standard technique described by Crawford. A total of 85 patients with severe congenital ptosis were enrolled in this study and submitted to surgical correction by frontalis suspension using autogenous fascia lata. Among these patients, 51 had previously undergone ptosis correction using other surgical techniques. The final lid level and contour were evaluated in addition to complications, such as lagophthalmos and ptosis recurrences. RESULTS: Overall, the final results were evaluated as good in 71 of the cases, whereas 11 cases were graded as satisfactory and three cases as poor according to the success criteria. The average increase in eyelid height, measured as the marginal reflex distance (MRD), was 2.9 mm. The best results were obtained in the group of patients with no previous eyelid surgeries. The examples of poor results could be attributed to lagophthalmos and were all confined to the group of patients with a previously failed surgery that employed synthetic suspension materials and levator shortening procedures. No recurrences were observed during the follow-up interval, which lasted an average of 6.4 months (range = 2-59 months). CONCLUSIONS: This technique allows a safe surgery with an overall high rate of success. This surgery is also safe and successful in cases of congenital ptosis with associated abnormalities, such as blepharophimosis, and in cases that require secondary ptosis repair.

Epithelial cells as alternative human biomatrices for comet assay.

The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

Does the adult human ciliary body epithelium contain "true" retinal stem cells?

Recent reports of retinal stem cells being present in several locations of the adult eye have sparked great hopes that they may be used to treat the millions of people worldwide who suffer from blindness as a result of retinal disease or injury. A population of proliferative cells derived from the ciliary body epithelium (CE) has been considered one of the prime stem cell candidates, and as such they have received much attention in recent years. However, the true nature of these cells in the adult human eye has still not been fully elucidated, and the stem cell claim has become increasingly controversial in light of new and conflicting reports. In this paper, we will try to answer the question of whether the available evidence is strong enough for the research community to conclude that the adult human CE indeed harbors stem cells.

Surgical approach to the superior mid-orbit.

Access to the superior mid-orbit is required for procedures on the levator muscle in the correction of upper eyelid ptosis and in surgery aimed at local lesions in this region. The purpose with this human cadaver study was to clarify the anatomical substrate for a surgical approach to the levator muscle and the upper mid-orbit structures, in which the orbital septum and the retroseptal fat pad is not harmed during surgery. Macro-anatomical dissections and histological examinations were performed on five human orbits from three formalin embalmed cadaver heads. It was found that the orbital septum extends posteriorly from its junction with the levator aponeurosis. This posterior continuation of the orbital septum encloses the superior orbital fat pad and separates this from the anterior surface of the levator muscle. In between the orbital septum and the levator, there is a dissection space that provides a minimal invasive access corridor to the structures in the upper mid-orbit.

DNA damage in lens epithelium of cataract patients in vivo and ex vivo.

PURPOSE: DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. METHODS: Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. RESULTS: DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. CONCLUSION: The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract.

Clinical transplantation of ex vivo expanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series.

PURPOSE: Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement. METHODS: We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye. RESULTS: Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11- to 28-month follow-up). CONCLUSIONS: Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter-species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.

Donor cornea transfer from Optisol GS to organ culture storage: a two-step procedure to increase donor tissue lifespan.

PURPOSE: Storage time for donor corneas in Optisol GS is limited compared to Eye Bank Organ Culture (EBOC). We here examine the epithelium on donor corneoscleral rims after primary storage in Optisol GS and subsequent incubation in EBOC. METHODS: Morphology was monitored by light and electron microscopy, expression of phenotypic and genotypic markers by immunohistochemistry and RT-PCR and changes in oxidative lipid and DNA damage by ELISA and COMET assay. RESULTS: A prominent loss of cells was observed after storage in Optisol GS. After maintenance in EBOC, spreading apical cells were Occludin(+) , while the staining for E-cadherin and Connexin-43 was less intense. There were an upregulation of Occludin and a downregulation of E-cadherin and Connexin-43. Eye Bank Organ Culture was associated with an ongoing proliferative activity and a downregulation of putative progenitor/stem cell marker ABCG2 and p63. Staining for 8-OHdG and Caspase-3 did not increase, while levels of malondialdehyde and number of DNA strand breaks and oxidized bases increased. CONCLUSIONS: This dual procedure should be pursued as an option to increase the storage time and the pool of available donor corneas. The observed downregulation of markers associated with stemness during EBOC is relevant considering the potential use of donor epithelium in the treatment of ocular surface disorders.

Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.

A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2 × 2 × 0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.

Oxidative stress gradient in a medium during human corneal organ culture.

PURPOSE: Lipid peroxidation content was measured in an organ culture medium after one-week storage of human donor corneas. Moreover, the effects of the medium on oxidative stress, antioxidant capacity, and the proliferation of cultured human corneal cells were studied. METHODS: The medium was sampled from the upper and lower halves of storage vials and from controls (n=42). Malondialdehyde (MDA) was measured by high pressure liquid chromatography (HPLC). Cultured human corneal epithelium (CRL-11515) was exposed to different medium samples and monitored for changes in MDA (enzyme-linked immunosorbent assay [ELISA]), total antioxidant capacity (antioxidant assay kit), and proliferation (Ki-67). RESULTS: A significant increase in MDA was observed in the organ culture medium in the lower level of storage vials. The addition of this fraction to cultured cells increased MDA significantly after 3 days, and the medium from both levels significantly increased MDA after 7 days. The medium from both levels significantly decreased the total antioxidant capacity of the cells but did not affect proliferative activity. CONCLUSIONS: An oxidative gradient with an evident biologic effect is established in the medium in vials during organ culture of human donor corneas. Donor tissue stored at the bottom or in lower levels of such vials is exposed to a significant amount of oxidative stress.

Corneal collagen crosslinking in vitro: inhibited regeneration of human limbal epithelial cells after riboflavin-ultraviolet-A exposure.

PURPOSE: To assess the hypothesis that during corneal crosslinking (CXL) treatment, riboflavin and ultraviolet-A (UVA) may have a toxic effect on human limbal epithelial cells. SETTING: Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ullevål, Oslo, Norway. DESIGN: Experimental study. METHODS: In this vitro study, limbal biopsies from corneoscleral rims collected after corneal transplantation were treated with the following combinations: riboflavin-UVA, riboflavin only, or UVA only; a control group received no treatment. After 3 weeks of cell culture, outgrowth of epithelium from the biopsies was evaluated by measuring the area of cell expansion and the number of cell layers. The explanted biopsies were analyzed for proliferation using immunohistochemistry marker Ki-67 and for apoptosis using the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) assay. RESULTS: The mean outgrowth from the biopsies was 2.25 mm(2) ± 6.90 (SD) in the riboflavin-UVA group, 181.4 ± 94.8 mm(2) in the riboflavin-only group, 128.5 ± 129.5 mm(2) in the UVA-only group, and 176.2 ± 114.0 mm(2) in the control group. There were no statistically significant between-group differences in the number of cell layers except in the riboflavin-UVA group, in which no cells were found. Detection of apoptosis with the TUNEL-assay was found in the riboflavin-UVA group only (4/5 sections). The proliferation marker Ki-67 was positive in some sections in all groups. CONCLUSION: Cytotoxicity and reduced cell expansion of human limbal epithelial cells occurred after riboflavin-UVA treatment in vitro, emphasizing the importance of avoiding riboflavin-UVA on the limbus during CXL.

Activation of neural progenitor cells in human eyes with proliferative vitreoretinopathy.

In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial - and NSC-associated markers both after direct fixation and repetitive passages. In conclusion, the adult human eye may harbor two different populations of neuroepithelial stem/progenitor cells; a non-glial population located in the proximal pars plana around peripheral cysts in addition to a population with Müller glia characteristics. Yet, we only found that the glial population was able to respond to retinal injury by targeted migration into the vitreous.

Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement.

In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM.