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Culture and characterization of oral mucosal epithelial cells on human amniotic membrane for ocular surface reconstruction.

Madhira SL, Vemuganti G, Bhaduri A, Gaddipati S, Sangwan VS, Ghanekar Y - Mol. Vis. (2008)

Bottom Line: Ultrastructural studies were also performed using electron microscopy.Electron microscopy demonstrated that the cells formed gap junctions and desmosomes.The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor.

View Article: PubMed Central - PubMed

Affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory,L.V. Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: To culture oral mucosal epithelial cells on deepithelialized human amniotic membrane without the use of feeder cells and to compare the characteristics of cultured oral cells with cultured limbal and conjunctival epithelial cells for use in ocular surface reconstruction.

Methods: Oral biopsies were obtained from healthy volunteers after informed consent and were cultured on deepithelialized amniotic membrane for three to four weeks. Confluent cultures of limbal, oral, and conjunctival cells were subjected to characterization of markers of stem cells and of epithelial differentiation by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry. Ultrastructural studies were also performed using electron microscopy.

Results: A sheet of healthy, stratified oral epithelial cells was obtained within three to four weeks of culture. Electron microscopy demonstrated that the cells formed gap junctions and desmosomes. RT-PCR analysis showed that cultured oral epithelial cells expressed markers of epithelial differentiation such as cytokeratin K3, K4, K13, K15 and connexin 43. The cells also expressed stem cell markers of epithelial cells such as DeltaN isoforms of p63 as well as p75, a marker for stem cells of oral epithelium. The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor.

Conclusions: Oral epithelial cells can be cultured as explants on deepithelialized amniotic membrane without using feeder cells. Characterization showed that these cells maintain the phenotypic characteristics of oral epithelial cells and that the culture is a heterogeneous population of differentiated cells and stem cells. We find the cultured oral epithelial cells usable for ocular surface reconstruction in patients having bilateral ocular surface diseases.

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Growth initiation and morphological characteristics of limbal, oral, and conjunctival cultures. A: Growth initiation from the three explant cultures is shown. B: Confluent cultures of limbal, oral, and conjunctival cells are shown. C: Hematoxylin and eosin staining of sections of limbal, oral, and conjunctival cultures are illustrated. D: PAS staining of the three explant cultures is also shown. The inset in conjunctival culture shows goblet cells detected in native conjunctival tissue.
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f1: Growth initiation and morphological characteristics of limbal, oral, and conjunctival cultures. A: Growth initiation from the three explant cultures is shown. B: Confluent cultures of limbal, oral, and conjunctival cells are shown. C: Hematoxylin and eosin staining of sections of limbal, oral, and conjunctival cultures are illustrated. D: PAS staining of the three explant cultures is also shown. The inset in conjunctival culture shows goblet cells detected in native conjunctival tissue.

Mentions: Oral, limbal, and conjunctival cells were cultured as explants on deepithelialized HAM using HCE medium. Compared to limbal and conjunctival cultures, cell growth initiation was slower in the oral cultures. Cell migration and growth initiated from oral explants within three to four days, which was later than that observed for limbal and conjunctival cultures (one to two days) as shown in Figure 1A. Healthy, confluent cultures of oral epithelial cells were obtained within three to four weeks (Figure 1B) as compared to 10–14 days in the case of limbal and conjunctival cultures. Cultured oral cells appeared to be slightly smaller in size compared to the cultured limbal or conjunctival cells. Even without airlifting, confluent oral epithelial cell cultures underwent stratification in places and formed two to three layers of cells as seen after hematoxylin-eosin (HE) staining (Figure 1C). No goblet cells were seen in these cultures by periodic acid Schiff (PAS) staining (Figure 1D).


Culture and characterization of oral mucosal epithelial cells on human amniotic membrane for ocular surface reconstruction.

Madhira SL, Vemuganti G, Bhaduri A, Gaddipati S, Sangwan VS, Ghanekar Y - Mol. Vis. (2008)

Growth initiation and morphological characteristics of limbal, oral, and conjunctival cultures. A: Growth initiation from the three explant cultures is shown. B: Confluent cultures of limbal, oral, and conjunctival cells are shown. C: Hematoxylin and eosin staining of sections of limbal, oral, and conjunctival cultures are illustrated. D: PAS staining of the three explant cultures is also shown. The inset in conjunctival culture shows goblet cells detected in native conjunctival tissue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254962&req=5

f1: Growth initiation and morphological characteristics of limbal, oral, and conjunctival cultures. A: Growth initiation from the three explant cultures is shown. B: Confluent cultures of limbal, oral, and conjunctival cells are shown. C: Hematoxylin and eosin staining of sections of limbal, oral, and conjunctival cultures are illustrated. D: PAS staining of the three explant cultures is also shown. The inset in conjunctival culture shows goblet cells detected in native conjunctival tissue.
Mentions: Oral, limbal, and conjunctival cells were cultured as explants on deepithelialized HAM using HCE medium. Compared to limbal and conjunctival cultures, cell growth initiation was slower in the oral cultures. Cell migration and growth initiated from oral explants within three to four days, which was later than that observed for limbal and conjunctival cultures (one to two days) as shown in Figure 1A. Healthy, confluent cultures of oral epithelial cells were obtained within three to four weeks (Figure 1B) as compared to 10–14 days in the case of limbal and conjunctival cultures. Cultured oral cells appeared to be slightly smaller in size compared to the cultured limbal or conjunctival cells. Even without airlifting, confluent oral epithelial cell cultures underwent stratification in places and formed two to three layers of cells as seen after hematoxylin-eosin (HE) staining (Figure 1C). No goblet cells were seen in these cultures by periodic acid Schiff (PAS) staining (Figure 1D).

Bottom Line: Ultrastructural studies were also performed using electron microscopy.Electron microscopy demonstrated that the cells formed gap junctions and desmosomes.The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor.

View Article: PubMed Central - PubMed

Affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory,L.V. Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: To culture oral mucosal epithelial cells on deepithelialized human amniotic membrane without the use of feeder cells and to compare the characteristics of cultured oral cells with cultured limbal and conjunctival epithelial cells for use in ocular surface reconstruction.

Methods: Oral biopsies were obtained from healthy volunteers after informed consent and were cultured on deepithelialized amniotic membrane for three to four weeks. Confluent cultures of limbal, oral, and conjunctival cells were subjected to characterization of markers of stem cells and of epithelial differentiation by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry. Ultrastructural studies were also performed using electron microscopy.

Results: A sheet of healthy, stratified oral epithelial cells was obtained within three to four weeks of culture. Electron microscopy demonstrated that the cells formed gap junctions and desmosomes. RT-PCR analysis showed that cultured oral epithelial cells expressed markers of epithelial differentiation such as cytokeratin K3, K4, K13, K15 and connexin 43. The cells also expressed stem cell markers of epithelial cells such as DeltaN isoforms of p63 as well as p75, a marker for stem cells of oral epithelium. The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor.

Conclusions: Oral epithelial cells can be cultured as explants on deepithelialized amniotic membrane without using feeder cells. Characterization showed that these cells maintain the phenotypic characteristics of oral epithelial cells and that the culture is a heterogeneous population of differentiated cells and stem cells. We find the cultured oral epithelial cells usable for ocular surface reconstruction in patients having bilateral ocular surface diseases.

Show MeSH
Related in: MedlinePlus