Viability of limbal epithelium after anterior lamellar harvesting using a microkeratome

Tulaya Tungsiripat, Melvin A. Sarayba, Mehran Taban, Paula M. Sweet, Kathryn E. Osann, Roy S. Chuck

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Objective: To determine the viability in cold eye bank storage of different layers of central and limbal corneal epithelium, including the limbal basal stem cell population, on days 0, 3, 6, and 9 after harvest using a large diameter microkeratome system. Methods: Twenty-two human whole globes not suitable for transplantation were obtained from an eye bank (San Diego Eye Bank, San Diego, California) and used for study. Large-diameter anterior corneal discs were prepared using a large diameter microkeratome and stained with calcein AM and an ethidium homodimer to differentiate live from dead cells, respectively. A laser confocal microscope and digital imaging were used to distinguish live (green) from dead (red) cells. Central and limbal epithelial regions were isolated and the middle and basal epithelial sections were cell counted by 3 independent observers. These sections were stored up to 9 days at 4°C in an eye bank corneal storage medium. Differences were tested using nonparametric methods. Main Outcome Measures: The percentage of live cells in each of these epithelial layers was determined for up to 9 days in cold eye bank corneal storage medium. Results: At all time points studied, the better protected basal epithelial layers displayed greater mean viability than the overlying middle epithelial layers. However, the difference was not statistically significant on all days. When comparing the basal epithelial viability of the limbal and central regions, after day 0 in 4°C cold organ culture, the observed viability of the limbal basal epithelium, the purported location of the limbal epithelial stem cell region, was significantly greater than that of the central epithelium. On day 0, median limbal basal versus central basal epithelial viability were 100% (range, 71.7-100%) versus 98.4% (range, 88.9-100%) (P>0.05); on day 3, 100% (range, 64.3-100%) versus 63.4% (range, 13.6-95.5%) (P<0.0005); on day 6, 95.0% (range, 35.0-100%) versus 28.0% (range, 0-92.0%) (P<0.0005); and on day 9, 95.0% (range, 3.7-100%) versus 68.6% (range, 0-100%) (P<0.0005). Conclusions: After microkeratome harvesting, the limbal basal epithelium is significantly longer lived in cold eye bank storage than central basal epithelium and the middle layers of limbal and central epithelium. This longevity not only bodes well for organ storage of limbal grafts, but also confirms the hardiness of the stem cell region.

Original languageEnglish (US)
Pages (from-to)469-475
Number of pages7
JournalOphthalmology
Volume111
Issue number3
DOIs
StatePublished - Mar 2004
Externally publishedYes

Fingerprint

Eye Banks
Epithelium
Stem Cells
Corneal Epithelium
Organ Culture Techniques
Lasers
Transplantation
Epithelial Cells
Outcome Assessment (Health Care)
Transplants
Population

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Viability of limbal epithelium after anterior lamellar harvesting using a microkeratome. / Tungsiripat, Tulaya; Sarayba, Melvin A.; Taban, Mehran; Sweet, Paula M.; Osann, Kathryn E.; Chuck, Roy S.

In: Ophthalmology, Vol. 111, No. 3, 03.2004, p. 469-475.

Research output: Contribution to journalArticle

Tungsiripat, Tulaya ; Sarayba, Melvin A. ; Taban, Mehran ; Sweet, Paula M. ; Osann, Kathryn E. ; Chuck, Roy S. / Viability of limbal epithelium after anterior lamellar harvesting using a microkeratome. In: Ophthalmology. 2004 ; Vol. 111, No. 3. pp. 469-475.
@article{3326bee6c1384250829dc37e229ab2a9,
title = "Viability of limbal epithelium after anterior lamellar harvesting using a microkeratome",
abstract = "Objective: To determine the viability in cold eye bank storage of different layers of central and limbal corneal epithelium, including the limbal basal stem cell population, on days 0, 3, 6, and 9 after harvest using a large diameter microkeratome system. Methods: Twenty-two human whole globes not suitable for transplantation were obtained from an eye bank (San Diego Eye Bank, San Diego, California) and used for study. Large-diameter anterior corneal discs were prepared using a large diameter microkeratome and stained with calcein AM and an ethidium homodimer to differentiate live from dead cells, respectively. A laser confocal microscope and digital imaging were used to distinguish live (green) from dead (red) cells. Central and limbal epithelial regions were isolated and the middle and basal epithelial sections were cell counted by 3 independent observers. These sections were stored up to 9 days at 4°C in an eye bank corneal storage medium. Differences were tested using nonparametric methods. Main Outcome Measures: The percentage of live cells in each of these epithelial layers was determined for up to 9 days in cold eye bank corneal storage medium. Results: At all time points studied, the better protected basal epithelial layers displayed greater mean viability than the overlying middle epithelial layers. However, the difference was not statistically significant on all days. When comparing the basal epithelial viability of the limbal and central regions, after day 0 in 4°C cold organ culture, the observed viability of the limbal basal epithelium, the purported location of the limbal epithelial stem cell region, was significantly greater than that of the central epithelium. On day 0, median limbal basal versus central basal epithelial viability were 100{\%} (range, 71.7-100{\%}) versus 98.4{\%} (range, 88.9-100{\%}) (P>0.05); on day 3, 100{\%} (range, 64.3-100{\%}) versus 63.4{\%} (range, 13.6-95.5{\%}) (P<0.0005); on day 6, 95.0{\%} (range, 35.0-100{\%}) versus 28.0{\%} (range, 0-92.0{\%}) (P<0.0005); and on day 9, 95.0{\%} (range, 3.7-100{\%}) versus 68.6{\%} (range, 0-100{\%}) (P<0.0005). Conclusions: After microkeratome harvesting, the limbal basal epithelium is significantly longer lived in cold eye bank storage than central basal epithelium and the middle layers of limbal and central epithelium. This longevity not only bodes well for organ storage of limbal grafts, but also confirms the hardiness of the stem cell region.",
author = "Tulaya Tungsiripat and Sarayba, {Melvin A.} and Mehran Taban and Sweet, {Paula M.} and Osann, {Kathryn E.} and Chuck, {Roy S.}",
year = "2004",
month = "3",
doi = "10.1016/j.ophtha.2003.06.012",
language = "English (US)",
volume = "111",
pages = "469--475",
journal = "Ophthalmology",
issn = "0161-6420",
publisher = "Elsevier Inc.",
number = "3",

}

TY - JOUR

T1 - Viability of limbal epithelium after anterior lamellar harvesting using a microkeratome

AU - Tungsiripat, Tulaya

AU - Sarayba, Melvin A.

AU - Taban, Mehran

AU - Sweet, Paula M.

AU - Osann, Kathryn E.

AU - Chuck, Roy S.

PY - 2004/3

Y1 - 2004/3

N2 - Objective: To determine the viability in cold eye bank storage of different layers of central and limbal corneal epithelium, including the limbal basal stem cell population, on days 0, 3, 6, and 9 after harvest using a large diameter microkeratome system. Methods: Twenty-two human whole globes not suitable for transplantation were obtained from an eye bank (San Diego Eye Bank, San Diego, California) and used for study. Large-diameter anterior corneal discs were prepared using a large diameter microkeratome and stained with calcein AM and an ethidium homodimer to differentiate live from dead cells, respectively. A laser confocal microscope and digital imaging were used to distinguish live (green) from dead (red) cells. Central and limbal epithelial regions were isolated and the middle and basal epithelial sections were cell counted by 3 independent observers. These sections were stored up to 9 days at 4°C in an eye bank corneal storage medium. Differences were tested using nonparametric methods. Main Outcome Measures: The percentage of live cells in each of these epithelial layers was determined for up to 9 days in cold eye bank corneal storage medium. Results: At all time points studied, the better protected basal epithelial layers displayed greater mean viability than the overlying middle epithelial layers. However, the difference was not statistically significant on all days. When comparing the basal epithelial viability of the limbal and central regions, after day 0 in 4°C cold organ culture, the observed viability of the limbal basal epithelium, the purported location of the limbal epithelial stem cell region, was significantly greater than that of the central epithelium. On day 0, median limbal basal versus central basal epithelial viability were 100% (range, 71.7-100%) versus 98.4% (range, 88.9-100%) (P>0.05); on day 3, 100% (range, 64.3-100%) versus 63.4% (range, 13.6-95.5%) (P<0.0005); on day 6, 95.0% (range, 35.0-100%) versus 28.0% (range, 0-92.0%) (P<0.0005); and on day 9, 95.0% (range, 3.7-100%) versus 68.6% (range, 0-100%) (P<0.0005). Conclusions: After microkeratome harvesting, the limbal basal epithelium is significantly longer lived in cold eye bank storage than central basal epithelium and the middle layers of limbal and central epithelium. This longevity not only bodes well for organ storage of limbal grafts, but also confirms the hardiness of the stem cell region.

AB - Objective: To determine the viability in cold eye bank storage of different layers of central and limbal corneal epithelium, including the limbal basal stem cell population, on days 0, 3, 6, and 9 after harvest using a large diameter microkeratome system. Methods: Twenty-two human whole globes not suitable for transplantation were obtained from an eye bank (San Diego Eye Bank, San Diego, California) and used for study. Large-diameter anterior corneal discs were prepared using a large diameter microkeratome and stained with calcein AM and an ethidium homodimer to differentiate live from dead cells, respectively. A laser confocal microscope and digital imaging were used to distinguish live (green) from dead (red) cells. Central and limbal epithelial regions were isolated and the middle and basal epithelial sections were cell counted by 3 independent observers. These sections were stored up to 9 days at 4°C in an eye bank corneal storage medium. Differences were tested using nonparametric methods. Main Outcome Measures: The percentage of live cells in each of these epithelial layers was determined for up to 9 days in cold eye bank corneal storage medium. Results: At all time points studied, the better protected basal epithelial layers displayed greater mean viability than the overlying middle epithelial layers. However, the difference was not statistically significant on all days. When comparing the basal epithelial viability of the limbal and central regions, after day 0 in 4°C cold organ culture, the observed viability of the limbal basal epithelium, the purported location of the limbal epithelial stem cell region, was significantly greater than that of the central epithelium. On day 0, median limbal basal versus central basal epithelial viability were 100% (range, 71.7-100%) versus 98.4% (range, 88.9-100%) (P>0.05); on day 3, 100% (range, 64.3-100%) versus 63.4% (range, 13.6-95.5%) (P<0.0005); on day 6, 95.0% (range, 35.0-100%) versus 28.0% (range, 0-92.0%) (P<0.0005); and on day 9, 95.0% (range, 3.7-100%) versus 68.6% (range, 0-100%) (P<0.0005). Conclusions: After microkeratome harvesting, the limbal basal epithelium is significantly longer lived in cold eye bank storage than central basal epithelium and the middle layers of limbal and central epithelium. This longevity not only bodes well for organ storage of limbal grafts, but also confirms the hardiness of the stem cell region.

UR - http://www.scopus.com/inward/record.url?scp=1442299747&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1442299747&partnerID=8YFLogxK

U2 - 10.1016/j.ophtha.2003.06.012

DO - 10.1016/j.ophtha.2003.06.012

M3 - Article

C2 - 15019321

AN - SCOPUS:1442299747

VL - 111

SP - 469

EP - 475

JO - Ophthalmology

JF - Ophthalmology

SN - 0161-6420

IS - 3

ER -