Elsevier

Experimental Eye Research

Volume 153, December 2016, Pages 122-132
Experimental Eye Research

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

https://doi.org/10.1016/j.exer.2016.09.012Get rights and content

Highlights

  • Effect of culture medium and carrier on ex vivo cultured human limbal epithelium.

  • Similar ultrastructure on transmission electron microscopy in all grafts.

  • Comparable keratin expression on immunohistochemistry in all grafts.

  • Similar microarray gene expression in cells cultured in the same medium.

Abstract

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).

Introduction

A healthy and transparent cornea is critically dependent on the continuous renewal of corneal epithelium by the limbal epithelial stem cells (LESCs). These stem cells are believed to be located in the basal layer of the limbus (Cotsarelis et al., 1989, Davanger and Evensen, 1971, Dua and Azuara-Blanco, 2000, Majo et al., 2008). Damage to the limbal area may result in loss or dysfunction of LESCs leading to limbal stem cell deficiency (LSCD). Clinically, LSCD is characterised by conjunctivalisation and neovascularisation of the corneal surface resulting in reduced corneal transparency and poor visual acuity, or even blindness (Tseng, 1996). In addition, patients with LSCD often experience recurrent epithelial defects and chronic inflammation, causing pain and photophobia with significantly reduced quality of life. In patients with LSCD, a vital and transparent corneal epithelium may be restored by grafting of limbal fragments (autologous or allogenic) or by the more recent procedure involving transplantation of ex vivo expanded limbal epithelial cells (LECs) (Pellegrini et al., 1997, Shortt et al., 2007, Tseng, 1996). Ex vivo expansion of LECs essentially involves the culture of limbal epithelial cells to form a confluent sheet of cells for transplantation to the ocular surface, either by explant culture, where limbal cells migrate out from a limbal tissue biopsy, or by suspension culture, in which limbal cells are cultured from a suspension of single cells prepared by enzymatic digestion of limbal tissue (Shortt et al., 2007). There is currently no consensus on a single optimal protocol for the ex vivo expansion of LECs, but rather several parallel protocols have evolved, with a crossover of different methodologies and techniques (Nakamura et al., 2004, Nakamura et al., 2006, Notara et al., 2007, Pellegrini et al., 1997, Rama et al., 2010, Tsai et al., 2000). Several clinics are now applying these techniques for treating LSCD patients (Ahmad et al., 2010, Pathak et al., 2013, Sangwan et al., 2006, Shortt et al., 2007). A complex medium (COM), including fetal bovine serum (FBS) and various animal-derived hormones and growth factors, is commonly used for the expansion process (Shortt et al., 2007). LECs have also been expanded using culture medium with human serum (HS) as the only growth supplement, without any animal-derived products (Di Girolamo et al., 2009, Nakamura et al., 2006, Sangwan et al., 2006). We have previously shown HS to be an attractive alternative to COM (Pathak et al., 2013, Shahdadfar et al., 2012) without the risk of transmission of pathogens associated with the use of animal-derived products (Schwab et al., 2006).

Significant variability in clinical outcome has been reported after transplantation of ex vivo expanded LECs, with success rates varying between 50 and 100% (Ahmad et al., 2010, Baylis et al., 2011, Rama et al., 2010, Shimazaki et al., 2007, Shortt et al., 2007). In our clinic, which is the only center in Scandinavia that offers this treatment, a success rate of 56% has been recorded (Pathak et al., 2013). The clinical outcome of transplantation may be affected by several factors, including type of initial ocular damage (Rama et al., 2010, Sangwan et al., 2006), ocular co-morbidity (DeSousa et al., 2009; Sangwan et al., 2006) and characteristics of the transplanted cells (Rama et al., 2010). The aim of the present study was to compare the characteristics of cells expanded ex vivo on human amniotic membrane (HAM) and on tissue-culture coated plastic polyester membrane inserts (PL) using either COM or HS, with possible implications for optimal ex vivo expansion of LECs and existing treatment strategies.

Section snippets

Materials and methods

The study was conducted in accordance with the Helsinki Declaration. All laboratory procedures and tissue harvesting were approved by the Local Committee for Medical Research Ethics. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise.

Ultrastructure of the expanded LECs using TEM

On TEM analysis, the ex vivo expanded tissue comprised of epithelial sheets of stratified cells in all four culture conditions (Fig. 1). The sheets consisted of 2–9 cell layers with superficial flattened cells and basal columnar/cuboidal cells attached to the HAM basement membrane or PL membrane. The LECs cultured in COM had more layers than those cultured in HS (Fig. 1, Table 1). Although all our cultures had numerous desmosomes, the LECs cultured in COM had more desmosomes than those cultured

Discussion

In this study, the properties of LECs expanded ex vivo in four different culture conditions were compared in order to better understand the factors that may influence clinical success rates in the treatment of patients with LSCD. Studies comparing various culture methods have shown that cell cultures in different media (Nakamura et al., 2006, Shahdadfar et al., 2012) result in LECs with similar features, although larger outgrowths and higher cell counts have been obtained in LECs cultured in HS

Conclusions

Ex vivo cultures of LECs express similar morphology and similar keratin staining patterns when cultured on HAM and PL in media supplemented with COM or HS. LECs cultured on HAM exhibited the presence of adhesion complexes between basal cells and HAM, which may improve the adherence between the expanded cells and the carrier. Microarray analysis revealed that gene expression was more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or

Author contributions

Conceived and designed the experiments: MP LD MM KJ BN AN. Performed the experiments: MP OKO NS SK KJ AN. Analysed the data: MP OKO SK KJ AN. Wrote the manuscript: MP AN KJ.

Acknowledgements

The authors have no financial or proprietary interests to disclose.

The research leading to these results has received funding from the Norwegian Financial Mechanism 2009–2014 and the Ministry of Education, Youth and Sports (Project Contract no. MSMT-28477/2014, project 7F14156); the Research Council of Norway; Blindemissionen IL; the Norwegian Association of the Blind and Partially Sighted; the Faculty of Medicine, University of Oslo, Oslo University Hospital; and the Norwegian Center for Stem

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