, Volume 70, Issue 2, pp 687–700 | Cite as

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

  • Mehmet Gürdal
  • Özlem Barut Selver
  • Kemal BaysalEmail author
  • İsmet Durak


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.


Limbal epithelial stem cells Limbal explant culture Gene expression Protein expression Autologous serum 



This study was supported by funding from Dokuz Eylul University, Research Grant No. 2013.KB.SAG.048 (KB) and The Scientific and Technological Research Council of Turkey; TUBITAK 1001 Research Grant No. 111S414 (ID).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Akinci MM, Turner H, Taveras M, Wolosin JM (2009) Differential gene expression in the pig limbal side population: implications for stem cell cycling, replication, and survival. Investig Ophthalmol Vis Sci 50:5630–5638. CrossRefGoogle Scholar
  2. Baylis O, Figueiredo F, Henein C, Lako M, Ahmad S (2011) 13 years of cultured limbal epithelial cell therapy: a review of the outcomes. J Cell Biochem 112:993–1002. CrossRefGoogle Scholar
  3. Bhattacharya R, Banerjee Mustafi S, Street M, Dey A, Dwivedi SKD (2015) Bmi-1: At the crossroads of physiological and pathological biology. Genes Dis 2:225–239. CrossRefGoogle Scholar
  4. Bray LJ, Heazlewood CF, Atkinson K, Hutmacher DW, Harkin DG (2012) Evaluation of methods for cultivating limbal mesenchymal stromal cells. Cytotherapy 14:936–947. CrossRefGoogle Scholar
  5. Budak MT, Alpdogan OS, Zhou M, Lavker RM, Akinci MM, Wolosin JM (2005) Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells. J Cell Sci 118:1715–1724. CrossRefGoogle Scholar
  6. de Paiva CS, Chen Z, Corrales RM, Pflugfelder SC, Li D-Q (2005) ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. Stem Cells 23:63–73. CrossRefGoogle Scholar
  7. Duan H, Wang Y, Yang L et al (2012) Pluripotin enhances the expansion of rabbit limbal epithelial stem/progenitor cells in vitro. Exp Eye Res 100:52–58. CrossRefGoogle Scholar
  8. Dziasko MA, Daniels JT (2016) Anatomical features and cell-cell ınteractions in the human limbal epithelial stem cell niche. Ocul Surf 14:322–330. CrossRefGoogle Scholar
  9. Figueira EC, Di Girolamo N, Coroneo MT, Wakefield D (2007) The phenotype of limbal epithelial stem cells. Investig Ophthalmol Vis Sci 48:144–156. CrossRefGoogle Scholar
  10. Ghoubay-Benallaoua D, Basli E, Goldschmidt P et al (2011) Human epithelial cell cultures from superficial limbal explants. Mol Vis 17:341–354Google Scholar
  11. González S, Deng SX (2013) Presence of native limbal stromal cells increases the expansion efficiency of limbal stem/progenitor cells in culture. Exp Eye Res 116:169–176. CrossRefGoogle Scholar
  12. Joe AW, Yeung SN (2014) Concise review: identifying limbal stem cells: classical concepts and new challenges. Stem Cells Transl Med 3:318–322. CrossRefGoogle Scholar
  13. Joseph A, Powell-Richards AOR, Shanmuganathan VA, Dua HS (2004) Epithelial cell characteristics of cultured human limbal explants. Br J Ophthalmol 88:393–398. CrossRefGoogle Scholar
  14. Jung S, Panchalingam KM, Rosenberg L, Behie LA (2012) Ex vivo expansion of human mesenchymal stem cells in defined serum-free media. Stem Cells Int 2012:123030. CrossRefGoogle Scholar
  15. Kameishi S, Umemoto T, Matsuzaki Y et al (2016) Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR. Biochem Biophys Res Commun 473:704–709. CrossRefGoogle Scholar
  16. Kawakita T, Shimmura S, Hornia A, Higa K, Tseng SCG (2008) Stratified epithelial sheets engineered from a single adult murine corneal/limbal progenitor cell. J Cell Mol Med 12:1303–1316. CrossRefGoogle Scholar
  17. Kinzebach S, Bieback K (2013) Expansion of mesenchymal stem/stromal cells under xenogenic-free culture conditions. Adv Biochem Eng Biotechnol 129:33–57. Google Scholar
  18. Kolli S, Lako M, Figueiredo F, Mudhar H, Ahmad S (2008) Loss of corneal epithelial stem cell properties in outgrowths from human limbal explants cultured on intact amniotic membrane. Regener Med 3:329–342. CrossRefGoogle Scholar
  19. Kolli S, Ahmad S, Lako M, Figueiredo F (2010) Successful clinical ımplementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency. Stem Cells 28:597–610. Google Scholar
  20. Kruse FE, Tseng SC (1992) Proliferative and differentiative response of corneal and limbal epithelium to extracellular calcium in serum-free clonal cultures. J Cell Physiol 151:347–360. CrossRefGoogle Scholar
  21. Levis H, Daniels JT (2009) New technologies in limbal epithelial stem cell transplantation. Curr Opin Biotechnol 20:593–597. CrossRefGoogle Scholar
  22. Liu Y, Ding Y, Ma P et al (2010) Enhancement of long-term proliferative capacity of rabbit corneal epithelial cells by embryonic stem cell conditioned medium. Tissue Eng Part C Methods 16:793–802. CrossRefGoogle Scholar
  23. Loureiro RR, Cristovam PC, Martins CM et al (2013) Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells. Mol Vis 19:69–77.
  24. Lužnik Z, Hawlina M, Maličev E et al (2016) Effect of cryopreserved amniotic membrane orientation on the expression of limbal mesenchymal and epithelial stem cell markers in prolonged limbal explant cultures. PLoS ONE 11:e0164408. CrossRefGoogle Scholar
  25. Mamo S, Gal AB, Polgar Z, Dinnyes A (2008) Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos. BMC Mol Biol 9:67. CrossRefGoogle Scholar
  26. O’Brien LE, Bilder D (2013) Beyond the niche: tissue-level coordination of stem cell dynamics. Annu Rev Cell Dev Biol 29:107–136. CrossRefGoogle Scholar
  27. Pathak M, Olstad OK, Drolsum L et al (2016) The effect of culture medium and carrier on explant culture of human limbal epithelium: a comparison of ultrastructure, keratin profile and gene expression. Exp Eye Res 153:122–132. CrossRefGoogle Scholar
  28. Pauklin M, Thomasen H, Pester A, Steuhl K-P, Meller D (2011) Expression of pluripotency and multipotency factors in human ocular surface tissues. Curr Eye Res 36:1086–1097. CrossRefGoogle Scholar
  29. Pellegrini G, Dellambra E, Golisano O et al (2001) P63 Identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98:3156–3161. CrossRefGoogle Scholar
  30. Pellegrini G, De Luca M, Arsenijevic Y (2007) Towards therapeutic application of ocular stem cells. Semin Cell Dev Biol 18:805–818. CrossRefGoogle Scholar
  31. Pellegrini G, Rama P, Di Rocco A, Panaras A, De Luca M (2014) Concise review: hurdles in a successful example of limbal stem cell-based regenerative medicine. Stem Cells 32:26–34. CrossRefGoogle Scholar
  32. Pieters T, Haenebalcke L, Hochepied T et al (2012) Efficient and user-friendly pluripotin-based derivation of mouse embryonic stem cells. Stem Cell Rev 8:768–778. CrossRefGoogle Scholar
  33. Qu Y, Chi W, Hua X et al (2015) Unique expression pattern and functional role of periostin in human limbal stem cells. PLoS ONE 10:e0117139. CrossRefGoogle Scholar
  34. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343. Accessed 13 April 2015
  35. Schermer A, Galvin S, Sun TT (1986) Differentiation-related expression of a major 64 K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 103:49–62. Accessed 05 June 2015
  36. Schlötzer-Schrehardt U, Kruse FE (2005) Identification and characterization of limbal stem cells. Exp Eye Res 81:247–264. CrossRefGoogle Scholar
  37. Selver OB, Barash A, Ahmed M, Wolosin JM (2011) ABCG2-dependent dye exclusion activity and clonal potential in epithelial cells continuously growing for 1 month from limbal explants. Investig Ophthalmol Vis Sci 52:4330–4337. CrossRefGoogle Scholar
  38. Seol D, Choe H, Zheng H et al (2011) Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc. BMC Res Notes 4:162. CrossRefGoogle Scholar
  39. Shahdadfar A, Haug K, Pathak M et al (2012) Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement. Exp Eye Res 97:1–9. CrossRefGoogle Scholar
  40. Shanmuganathan VA, Foster T, Kulkarni BB et al (2007) Morphological characteristics of the limbal epithelial crypt. Br J Ophthalmol 91:514–519. CrossRefGoogle Scholar
  41. Sharma RR, Pollock K, Hubel A, McKenna D (2014) Mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices. Transfusion 54:1418–1437. CrossRefGoogle Scholar
  42. Szabó DJ, Noer A, Nagymihály R et al (2015) Long-term cultures of human cornea limbal explants form 3D structures ex vivo—implications for tissue engineering and clinical applications. Ljubimov AV (ed) PLoS One 10:e0143053.
  43. Tseng SC (1989) Concept and application of limbal stem cells. Eye (Lond) 3:141–157. CrossRefGoogle Scholar
  44. Tseng SCG, Chen S-Y, Shen Y-C, Chen W-L, Hu F-R (2010) Critical appraisal of ex vivo expansion of human limbal epithelial stem cells. Curr Mol Med 10:841–850. Accessed 6 June 2015
  45. Umemoto T, Yamato M, Nishida K, Yang J, Tano Y, Okano T (2006) Limbal epithelial side-population cells have stem cell-like properties, including quiescent state. Stem Cells 24:86–94. CrossRefGoogle Scholar
  46. Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034. Accessed 27 July 2014
  47. Wang D-Y, Hsueh Y-J, Yang VC, Chen J-K (2003) Propagation and phenotypic preservation of rabbit limbal epithelial cells on amniotic membrane. Investig Ophthalmol Vis Sci 44:4698–4704. Accessed 12 Dec 2014
  48. Xie H-T, Chen S-Y, Li G-G, Tseng SCG (2011) Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation. Stem Cells 29:1874–1885. CrossRefGoogle Scholar
  49. Yoon JJ (2014) Limbal stem cells: central concepts of corneal epithelial homeostasis. World J Stem Cells 6:391. CrossRefGoogle Scholar
  50. Yoon J, Ismail S, Sherwin T (2014) Limbal stem cells: central concepts of corneal epithelial homeostasis. World J Stem Cells 6:391–403. CrossRefGoogle Scholar
  51. Zhao Y, Ma L (2015) Systematic review and meta-analysis on transplantation of ex vivo cultivated limbal epithelial stem cell on amniotic membrane in limbal stem cell deficiency. Cornea 34:592–600. CrossRefGoogle Scholar

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© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Biochemistry, Medical FacultyDokuz Eylul Universityİnciraltı, İzmirTurkey
  2. 2.Department of Ophthalmology, Medical FacultyDokuz Eylul Universityİnciraltı, İzmirTurkey
  3. 3.Department of Ophthalmology, Medical FacultyEge UniversityBornova, İzmirTurkey

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