Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro

Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla

doi:10.1007/s00417-015-3235-4

Cited by 10 publications

(6 citation statements)

References 32 publications

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“…However, cells retain the ability to proliferate in vitro when stimulated with serum and growth factors 9 , 10 . Indeed, epidermal growth factor (EGF) 11 , basic fibroblast growth factor (bFGF) 12 , 13 and various sera and media formulations have been reported to enable cell expansion of CECs 14 – 17 . However, CECs can change morphology after repeated passaging, going from an endothelial phenotype (monolayer of hexagonal cells) to a fibroblastic-like phenotype (elongated cells that grow on top of each other).…”

Section: Introductionmentioning

confidence: 99%

TGF-β1 promotes cell barrier function upon maturation of corneal endothelial cells

Leclerc

Roy

Santerre

et al. 2018

Sci Rep

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Human corneal endothelial cells (HCECs) easily become fibroblastic-like when cultured, rendering them unsuitable for tissue engineering of the cornea. Transforming growth factor β (TGF-β) could be a key factor in this phenomenon; however, TGF-β is also known to maintain the endothelium in a quiescent state in vivo. This work aimed to compare the effects of TGF-β1 on the phenotype of HCECs during the proliferation and maturation phases. Our results show that addition of TGF-β1 during the active proliferation phase produced fibroblastic HCECs and loss of the cell junction markers ZO-1 and n-cadherin, independent from the presence of epidermal growth factor (EGF). By contrast, addition of TGF-β1 in maturation media containing few mitogens led to an endothelial phenotype and functional cell junctions as HCECs developed a high trans-endothelial resistance. Furthermore, addition of AG-1478, an epithelial growth factor receptor inhibitor, enhanced the gain of the endothelial phenotype and cell barrier function. Overall, these results show that TGF-β1 can be used to promote the formation of a typical leaky endothelial barrier during the maturation phase of cultured HCECs. A two-phase culture of HCECs using distinct proliferation and maturation media could also be key for developing ideal HCEC culture conditions.

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Section: Introductionmentioning

confidence: 99%

TGF-β1 promotes cell barrier function upon maturation of corneal endothelial cells

Leclerc

Roy

Santerre

et al. 2018

Sci Rep

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“…Logically, frequent medium change, with repeat cell stress, may also result in the loss of LECs 44 . Interestingly, Valtink et al observed that corneal epithelial cells may be more susceptible to culture medium composition than corneal endothelial cells 45 . In addition, potential pH changes may also play an important role in corneal LEC loss in culture, which could also be studied in the future.…”

Section: Discussionmentioning

confidence: 99%

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

Böhringer

Stachon

et al. 2023

Klin Monbl Augenheilkd

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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: Three-hundred and four corneoscleral rings of 215 donors have been 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 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 KSFM 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 (360º, 270º 180º, 120º, 90º, less than 90º) (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), 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 culture, in vitro. Keywords: limbal epithelial cell culture, organ culture, stepwise linear regression algorithm ABSTRAKT Hintergrund: Um verschiedene potentielle 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 dreihundertvier 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 mit Hilfe 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 (360º, 270º 180º, 120º, 90º, weniger als 90º) (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 ‧ Größe des korneoskleralen Rings (p=0,007). Schlussfolgerungen: Der Erfolg der LEC Kultivierung steigt mit dem 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. Schlüsselwörter: Limbale Epithelzellkultur, Organkultur, schrittweiser linearer Regressionsalgorithmus

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“…In addition, different media and supplements applied might also influence the ECD loss (Valtink et al. ).…”

Section: Discussionmentioning

confidence: 99%

Gebauer SLc Original and Moria One‐Use Plus automated microkeratomes for ultrathin Descemet's stripping automated endothelial keratoplasty preparation

Fuest

Salla

Hermel

et al. 2016

Acta Ophthalmologica

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Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro

Abstract: The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

Cited by 10 publications

References 32 publications

TGF-β1 promotes cell barrier function upon maturation of corneal endothelial cells

TGF-β1 promotes cell barrier function upon maturation of corneal endothelial cells

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

Gebauer SLc Original and Moria One‐Use Plus automated microkeratomes for ultrathin Descemet's stripping automated endothelial keratoplasty preparation

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