Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro – A Stepwise Linear Regression Algorithm

Abstract

Purpose To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm.

Methods Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO₂ atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm.

Results The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007).

Conclusions The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro.

Zusammenfassung

Hintergrund Um verschiedene potenzielle Faktoren auf das Wachstum humaner limbaler Epithelzellen (LEC) aus Hornhautspendergewebe nach Langzeitlagerung (Organkultur) in vitro zu untersuchen, wurde ein schrittweiser linearer Regressionsalgorithmus verwendet.

Methoden Für unsere Experimente wurden 304 Hornhautringe von 215 Spendern verwendet. Zunächst wurden die Gewebestücke mit 4,0 mg/mL Kollagenase A bei 37 °C mit 95% relativer Luftfeuchtigkeit und 5% CO₂-Atmosphäre über Nacht inkubiert. Danach wurden die limbalen Epithelzellen mithilfe eines 20 µm CellTricks-Filters von den limbalen Keratozyten getrennt. Die humanen LECs wurden in KSFM-Medium, 1% Penicillin/Streptomycin (P/S), 0,02% epidermalem Wachstumsfaktor (EGF) und 0,3% Rinderhypophysenextrakt (BPE) kultiviert. Der potenzielle Einfluss des Spenderalters (Kovariate), der postmortalen Zeit (Kovariate), des Mediums (Kovariate), der Größe des verwendeten korneoskleralen Rings (360°, 270°180°, 120°, 90°, weniger als 90°; Kovariate), der Endothelzelldichte (ECD; Kovariate), Geschlecht (Faktor), Anzahl der Kulturmediumwechsel während der Organkultur (Faktor) und Herkunft des Spenders (spendende Institution und aufbewahrende Institution, Faktor) auf das Wachstum der LEC wurde mit einem schrittweisen linearen Regressionsalgorithmus analysiert.

Ergebnisse Die Rate der erfolgreichen Kultivierung humaner LEC betrug 37,5%. Die mit dem schrittweisen linearen Regressionsalgorithmus berechneten relevanten Einflussparameter auf das LEC-Wachstum waren: Intercept (p < 0,001), Spenderalter (p = 0,002), Anzahl der Kulturmediumwechsel während der Organkultur (p < 0,001), Gesamtmediumzeit (p = 0,181), Größe des verwendeten korneoskleralen Rings (p = 0,007) sowie Mediumzeit und Größe des korneoskleralen Rings (p = 0,007).

Schlussfolgerungen Der Erfolg der LEC-Kultivierung steigt mit abnehmendem Alter des Spenders, der geringeren Anzahl von Wechseln des Organkulturmediums während der Lagerung, der kürzeren Mediendauer in der Organkultur und der kleineren Größe des korneoskleralen Rings. Dieser von uns berechneter schrittweiser linearer Regressionsalgorithmus kann uns bei der Optimierung der LEC-Kultur in vitro helfen.

Conclusion Box

Already known:

• Due to the limited availability of fresh ocular tissue and the logistic advantages of stored tissue, corneoscleral rings have been used to generate limbal epithelial cell (LEC) cultures for transplantation, usually as explant cultures. Nevertheless, the effect of potential factors on LEC outgrowth in vitro has not been previously analyzed using a stepwise linear regression algorithm.

• Our present work assessed various potential factors on human LEC outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm.

Newly described:

• The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size.

• Our stepwise linear regression algorithm may help us in optimizing LEC culture in vitro.

Key words

limbal epithelial cell culture - organ culture - stepwise linear regression algorithm

Schlüsselwörter

limbale Epithelzellkultur - Organkultur - schrittweiser linearer Regressionsalgorithmus

Publikationsverlauf

Eingereicht: 17. November 2022

Angenommen: 02. Mai 2023

Accepted Manuscript online:
02. Mai 2023

Artikel online veröffentlicht:
19. Oktober 2023

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