title: Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells.
abstract: OBJECTIVE
  To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells.
  
  
  METHODS
  Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5'-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT-PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining.
  
  
  RESULTS
  Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM.
  
  
  CONCLUSIONS
  Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM.
authors: Ocular Surface Advanced Center, Federal University of São Paulo, São Paulo, Brazil.
authors: Renata Ruoco
authors: RR
authors: Loureiro
authors: Priscila Cardoso
authors: PC
authors: Cristovam
authors: Caio Marques
authors: CM
authors: Martins
authors: Joyce Luciana
authors: JL
authors: Covre
authors: Juliana Aparecida
authors: JA
authors: Sobrinho
authors: José Reinaldo da Silva
authors: JR
authors: Ricardo
authors: Rossen Myhailov
authors: RM
authors: Hazarbassanov
authors: Ana Luisa
authors: AL
authors: Höfling-Lima
authors: Rubens
authors: R
authors: Belfort
authors: Mauro
authors: M
authors: Nishi
authors: José Álvaro Pereira
authors: JÁ
authors: Gomes
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media; D016895:Culture Media, Serum-Free; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D053558:Keratin-3 / Q000235:genetics / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D003470:Culture Media; D016895:Culture Media, Serum-Free; C479360:KRT3 protein, human; D053558:Keratin-3; D009363:Neoplasm Proteins; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins
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