Comparative study of substrate free and amniotic membrane scaffolds for cultivation of limbal epithelial sheet

• Published in: Scientific reports 2018-10, Vol.8 (1), p.14628-11, Article 14628
• Main Authors: Lee, Hyun Jung, Nam, Sang Min, Choi, Sae Kyung, Seo, Kyoung Yul, Kim, Hyun Ok, Chung, So-Hyang
• Format: Article
• Language: English
• Subjects: 101/28; 13/100; 13/106; 14/19; 631/136/532/489; 692/308/2171; Amniotic membrane; Animal models; Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2 - metabolism; ATP-binding Cassette Sub-family G Member 2 (ABCG2); Cell size; Comparative studies; Cornea; Corneal Transplantation; Cultivation; Culture media; Culture Media, Serum-Free - chemistry; Cytokeratin; Epithelium, Corneal - growth & development; Explants; Humanities and Social Sciences; Humans; Investigations; Keratin-15 - metabolism; Limbal Explants; Limbal Stem Cell Deficiency (LSCD); Limbus Corneae - growth & development; Low JC; Medicine; multidisciplinary; Neoplasm Proteins - metabolism; Outgrowth Sheets; Progenitor cells; Rabbits; Science; Science (multidisciplinary); Stem cells; Tissue Culture Techniques - methods; Tissue Scaffolds - chemistry; Transcription Factors - metabolism; Transplantation; Tumor Suppressor Proteins - metabolism
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-32914-0

Transplantation of cultivated limbal epithelial transplantation has been proven to restore the corneal surface in limbal stem cell deficiency (LSCD). Here we comparatively investigated the optimized conditions and the efficiency of limbal epithelial sheet growth in three media conditions as well as with substrate free (transwell), human amniotic membrane (HAM) sutured onto transwell inserts (HAMTW), and HAM slide scaffold (HAMS). Outcomes evaluated were outgrowth sheet size from limbal explants, expression of stem/progenitor cell markers p63α, ABCG2 and CK15, and colony formation efficiency (CFE). Additionally, limbal epithelial sheets on HAMS were transplanted into corneas of LSCD rabbit models. Limbal epithelial sheets with 5% human AB serum showed the greatest increase in ABCG2 efflux activity (JC1 low ), p63α expression, and CFE compared in both conditions without HAM and with HAM, respectively. The outgrowth sheet size, cell yield, and Ki67 expression were increased in limbal epithelial sheets on HAMS compared to transwell and HAMTW. ABCG2 efflux activity, p63α and CK15 expressions, and CFE were also increased in limbal epithelial sheets on HAMS as well. In corneas of transplanted rabbit LSCD models, p63α expressions were noted in the basal layers and CK12 expressions were observed in superficial layers. Cultivation of limbal epithelial sheet on HAMS with xeno-free medium enhances the growth and stemness of limbal epithelial sheets.