J Korean Med Sci.  2008 Oct;23(5):864-869. 10.3346/jkms.2008.23.5.864.

Efficient Cultivation Conditions for Human Limbal Epithelial Cells

• Kim MK
• Lee JL
• Oh JY
• Shin MS
• Shin KS
• Wee WR
• Lee JH
• Park KS
• Son YS

• KMID: 1783074
• DOI: http://doi.org/10.3346/jkms.2008.23.5.864

Abstract

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding casette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

Keyword

Amniotic Membrane; Cornea; Limbal Epithelial Cell; CK12; p63

MeSH Terms

3T3 Cells
Animals
Cell Culture Techniques/*instrumentation/*methods
Cells, Cultured
Cytological Techniques
DNA Primers/chemistry
Epithelial Cells/*metabolism
Humans
Immunohistochemistry/methods
Keratin-12/metabolism
Mice
Models, Biological
Phosphoproteins/metabolism
Reverse Transcriptase Polymerase Chain Reaction
Stem Cells/cytology
Trans-Activators/metabolism

Figure

• 

  Fig. 1 Cultivated limbal epithelial cells on amniotic membranes (AMs) for 11 days. The limbal epithelial cells on cryopreserved AM (A) or freeze-dried AM (B) were slowly growing, compared with those with 3T3 feeders (C). Original magnification ×200. (D) RT-PCR results in limbal epithelial cells cultivated on cryopreserved (lane A) and freeze-dried amniotic membrane (lane B) or with 3T3 feeders (lane C). (E) The expression of all of them did not show any differences in cultivated epithelial cells between on cryo-preserved amniotic membrane and freeze-dried amniotic membrane. (F) The relative expression of mRNA for P63 and cytokeratin 12 was significantly lower in epithelial cells cultivated with amniotic membrane including both cryo-preserved and dried-freeze AM (p=0.028 for p63, 0.004 for cytokeratin 12, Mann Whitney U test) than that of 3T3-cocultured cells. Conx 43, connexin 43; CK 12, cytokeratin 12. Arrow means the location of band for ABCG2 (110 bp).

• 

  Fig. 2 Immunocytochemistry for p63 in cultivated limbal cells on the cryopreserved amniotic membrane (A) and 3T3 feeder payer (C). Low expression of p63 was noted in the cells cultured on AM. (B, D) Hoechst counterstaining.

• 

  Fig. 3 Immunocytochemistry for cytokeratin 12 (CK12) in cultivated limbal cells on the cryopreserved amniotic membrane (A) and 3T3 feeder payer (C). Low expression of CK12 was noted in the cells cultured on AM. (B, D) Hoechst counterstaining.

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