Fine Structure and Collagen Synthesis Activity of Monolayer Cultures of Rabbit Corneal Endothelium

Perlman, Marcia; Baum, Jules; Kaye, Gordon I.

doi:10.1083/jcb.63.1.306

Cited by 31 publications

(4 citation statements)

References 17 publications

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“…Procollagen I concentration was six times higher in CECs than in the control cells. This is in accordance with a previous study that reports CECs higher ability of proline hydroxylation than most of the fibroblastic cell lines [31]. The Na/K-ATPase colorimetric analysis for CECs is a low cost and feasible methodology that results useful to screen testing.…”

Section: Cell Yieldsupporting

confidence: 92%

Experimental design of a culture approach for corneal endothelial cells

Montalvo-Parra

Vidal-Paredes

Calzada-Rodríguez

et al. 2020

Preprint

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Background The harvesting of corneal endothelial cells (CECs) has received special attention given its potential as therapy for corneal blindness. The main challenges to overcome for this purpose are related to the culture media formulation, cellular density at the primary isolation, and the number of passages in which CECs can retain their functional characteristics. The alternance of different media formulations to harvest CECs has an impact on the cellular yield and morphology. We herein analyzed eight different sequences of growth factor-supplemented proliferative (P) and non-supplemented resting (R) media upon passages to find the optimal P/R culture media sequence in regards of cell yield, morphology, procollagen I production, ATPase function, and the expression of ZO-1 and ATPase. Results PRPR and PRRR sequences showed the higher cell yield and hexagonal morphology rate. CECs cultured in the PRRR sequence produced procollagen I, showed Na/K-ATPase function, and expression of ZO-1 and Na/K-ATPase by immunocytochemistry. Our study sets a culture approach to guarantee CECs expansion, as well as functionality for their potential use in tissue engineering and in vivo analyses. Conclusions Alternation of P and R culture media improves CECs culture. PRRR sequence demonstrated to be effective and for CECs proliferation lowering the cost implied in PRPR sequences. We discarded the use of pituitary extract and ROCK inhibitors as essential for CECs proliferation.

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Section: Cell Yieldsupporting

confidence: 92%

Experimental design of a culture approach for corneal endothelial cells

Montalvo-Parra

Vidal-Paredes

Calzada-Rodríguez

et al. 2020

Preprint

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show abstract

“…Collagen I is one of the most abundant proteins produced by the corneal endothelium [38] whose arrangement is involved in corneal clarity. Procollagen I concentration was six times higher in CEC than in the control cells compared with a previous study that reports CEC higher ability of proline hydroxylation than most of the fibroblastic cell lines [39]. The Na þ /K þ -ATPase colorimetric analysis for CEC is a low cost and feasible methodology that results in useful to screen testing.…”

Section: Cell Yield and Morphologymentioning

confidence: 58%

Experimental design of a culture approach for corneal endothelial cells of New Zealand white rabbit

Montalvo-Parra

Vidal-Paredes

Calzada-Rodríguez

et al. 2020

Heliyon

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The harvesting of corneal endothelial cells (CEC) has received special attention due to its potential as a therapy for corneal blindness. The main challenges are related to the culture media formulation, cellular density at the primary isolation, and the number of passages in which CEC can retain their functional characteristics. To alternate different media formulations to harvest CEC has an impact on the cellular yield and morphology. Therefore, we analyzed four different sequences of growth factor-supplemented Stimulatory (S) and non-supplemented Quiescent (Q) media, upon passages to find the optimal S-Q culture sequence. We assessed cell yield, morphology, procollagen I production, Na + /K + -ATPase function, and the expression of ZO-1 and Na + /K + -ATPase. Our results show SQSQ and SQQQ sequences with a balance between an improved cell yield and hexagonal morphology rate. CEC cultured in the SQQQ sequence produced procollagen I, showed Na + /K + -ATPase function, and expression of ZO-1 and Na + /K + -ATPase. Our study sets a culture approach to guarantee CEC expansion, as well as functionality for their potential use in tissue engineering and in vivo analyses. Thus, the alternation of S and Q media improves CEC culture. SQQQ sequence demonstrated CEC proliferation and lower the cost implied in SQSQ sequences. We discarded the use of pituitary extract and ROCK inhibitors as essential for CEC proliferation.

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“…The posterior surface of the cornea is lined by a monolayer of cells, termed the endothelium, which rests * Correspondence and reprints upon Descemet's membrane (DM), the basement membrane. These cells are responsible for DM growth and maintenance in vivo and secrete basement membrane-like components in vitro [43,48]. Immunocytochemicai investigations on tissue sections from some species [35,49], including man [52], have demonstrated the presence of FN at the stromal but not the cellular side of DM.…”

Section: Introductionmentioning

confidence: 99%

Ultrastructural immunocytochemical localization of fibronectin deposition during corneal endothelial wound repair. Evidence for cytoskeletal involvement

Sabet

Gordon

1989

Biology of the Cell

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The distribution of the extracellular matrix (ECM) protein, fibronectin (FN), has been examined ultrastructurally in noninjured and injured rat corneal endothelium in vivo and in vitro by immunoperoxidase cytochemistry. In noninjured endothelia, FN was observed within the rough endoplasmic reticulum (RER) cisternae but not along the cell-Descemet's membrane (DM) interface. Twenty-four and 48 h after a circular freeze injury, immunoperoxidase reaction product was detected at the cell-DM interface as well as within cytoplasmic vesicles and intercellular spaces. By 1 and 2 wk post-injury, a line of reaction product could still be demonstrated at the cell-DM interface and evidence for newly deposited basement membrane material was observed in this region. In order to understand whether fibronectin deposition during wound repair was dependent on cytoskeletal influences, organ culture experiments were performed in which the media was supplemented with either 10(-8) M colchicine or 2.5 X 10(-3) M cytochalasin B. Without inhibitors, injured corneas cultured for 24 h had FN deposition at the cell-DM interface similar to the in vivo results. Corneas cultured in the presence of cytochalasin B also showed FN deposition at the cell-DM interface. However, when injured endothelia were cultured in the presence of colchicine, no reaction product was observed at the cell-DM interface, although it could be detected intracellularly within RER. Incubating the tissues in the presence of puromycin abolished all extracellular and intracellular staining. These results indicate that during wound repair, corneal endothelial cells produce fibronectin and deposit it upon Descemet's membrane by a mechanism that may be mediated by microtubules.

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Fine Structure and Collagen Synthesis Activity of Monolayer Cultures of Rabbit Corneal Endothelium

Cited by 31 publications

References 17 publications

Experimental design of a culture approach for corneal endothelial cells

Experimental design of a culture approach for corneal endothelial cells

Experimental design of a culture approach for corneal endothelial cells of New Zealand white rabbit

Ultrastructural immunocytochemical localization of fibronectin deposition during corneal endothelial wound repair. Evidence for cytoskeletal involvement

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