Human Keratocytes Cultured on Amniotic Membrane Stroma Preserve Morphology and Express Keratocan

Edgar M. Espana, Hua He, Tetsuya Kawakita, Mario A. Di Pascuale, Vadrevu K. Raju, Chia Yang Liu, Scheffer C G Tseng

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

PURPOSE. To develop a new method of expanding human corneal keratocytes in serum while maintaining their characteristic morphology and keratocan expression. METHODS. Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/mL of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. On confluence, cells on AM were continuously subcultured for six passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were reseeded on AM. Cellular morphology and cell-cell networks were assessed by phase-contrast microscopy and a cell viability assay, respectively. Expression of keratocan was determined by RT-PCR and Western blot analysis. RESULTS. Trephined stroma yielded 91,600 ± 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM, even in 10% FBS but fibroblastic on plastic. Such a difference in morphology correlated with expression of keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM, even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passages 3 and 11 did not reverse their morphology or synthesize keratocan when reseeded on plastic in 1% FBS or on AM. CONCLUSIONS. Human keratocytes maintain their characteristic morphology and keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.

Original languageEnglish
Pages (from-to)5136-5141
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number12
DOIs
Publication statusPublished - 2003 Dec
Externally publishedYes

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Amnion
Plastics
Corneal Keratocytes
Western Blotting
Corneal Stroma
Phase-Contrast Microscopy
Polymerase Chain Reaction
Collagenases
Serum
Dendritic Cells
Digestion
Cultured Cells
Cell Survival
Fibroblasts

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Espana, E. M., He, H., Kawakita, T., Di Pascuale, M. A., Raju, V. K., Liu, C. Y., & Tseng, S. C. G. (2003). Human Keratocytes Cultured on Amniotic Membrane Stroma Preserve Morphology and Express Keratocan. Investigative Ophthalmology and Visual Science, 44(12), 5136-5141. https://doi.org/10.1167/iovs.03-0484

Human Keratocytes Cultured on Amniotic Membrane Stroma Preserve Morphology and Express Keratocan. / Espana, Edgar M.; He, Hua; Kawakita, Tetsuya; Di Pascuale, Mario A.; Raju, Vadrevu K.; Liu, Chia Yang; Tseng, Scheffer C G.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 12, 12.2003, p. 5136-5141.

Research output: Contribution to journalArticle

Espana, EM, He, H, Kawakita, T, Di Pascuale, MA, Raju, VK, Liu, CY & Tseng, SCG 2003, 'Human Keratocytes Cultured on Amniotic Membrane Stroma Preserve Morphology and Express Keratocan', Investigative Ophthalmology and Visual Science, vol. 44, no. 12, pp. 5136-5141. https://doi.org/10.1167/iovs.03-0484
Espana, Edgar M. ; He, Hua ; Kawakita, Tetsuya ; Di Pascuale, Mario A. ; Raju, Vadrevu K. ; Liu, Chia Yang ; Tseng, Scheffer C G. / Human Keratocytes Cultured on Amniotic Membrane Stroma Preserve Morphology and Express Keratocan. In: Investigative Ophthalmology and Visual Science. 2003 ; Vol. 44, No. 12. pp. 5136-5141.
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AU - Espana, Edgar M.

AU - He, Hua

AU - Kawakita, Tetsuya

AU - Di Pascuale, Mario A.

AU - Raju, Vadrevu K.

AU - Liu, Chia Yang

AU - Tseng, Scheffer C G

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N2 - PURPOSE. To develop a new method of expanding human corneal keratocytes in serum while maintaining their characteristic morphology and keratocan expression. METHODS. Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/mL of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. On confluence, cells on AM were continuously subcultured for six passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were reseeded on AM. Cellular morphology and cell-cell networks were assessed by phase-contrast microscopy and a cell viability assay, respectively. Expression of keratocan was determined by RT-PCR and Western blot analysis. RESULTS. Trephined stroma yielded 91,600 ± 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM, even in 10% FBS but fibroblastic on plastic. Such a difference in morphology correlated with expression of keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM, even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passages 3 and 11 did not reverse their morphology or synthesize keratocan when reseeded on plastic in 1% FBS or on AM. CONCLUSIONS. Human keratocytes maintain their characteristic morphology and keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.

AB - PURPOSE. To develop a new method of expanding human corneal keratocytes in serum while maintaining their characteristic morphology and keratocan expression. METHODS. Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/mL of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. On confluence, cells on AM were continuously subcultured for six passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were reseeded on AM. Cellular morphology and cell-cell networks were assessed by phase-contrast microscopy and a cell viability assay, respectively. Expression of keratocan was determined by RT-PCR and Western blot analysis. RESULTS. Trephined stroma yielded 91,600 ± 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM, even in 10% FBS but fibroblastic on plastic. Such a difference in morphology correlated with expression of keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM, even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passages 3 and 11 did not reverse their morphology or synthesize keratocan when reseeded on plastic in 1% FBS or on AM. CONCLUSIONS. Human keratocytes maintain their characteristic morphology and keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.

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