Characterization of cryopreserved primary human corneal endothelial cells cultured in human serum-supplemented media

Vianna, Lucas Monferrari Monteiro; Li, Hao-Dong; Holiman, Jeffrey D.; Stoeger, Christopher G.; Belfort, Rubens; Jun, Albert S.

doi:10.5935/0004-2749.20160011

Cited by 8 publications

(5 citation statements)

References 16 publications

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“…We had previously tried to culture primary CEnCs within a serum-free environment, replacing FBS with a KnockOut Serum Replacer (KO-SR), or removing FBS altogether, but isolated primary CEnCs were unable to thrive in those conditions (results not shown). Hence, with some form of serum being necessary, we continued its use, and for this study compared our current FBS with sera from two other sources, another bovine serum, Equafetal, and commercially available HS, reported to support the culture of primary CEnCs 40 , 41 . Interestingly, in our comparisons, primary CEnCs cultured in either of the two bovine sera were comparable as opposed to the morphological and proliferative inconsistency observed for CEnCs from all 5 donors that were propagated in HS supplemented medium (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Being able to cryo-preserve primary human CEnCs is important as it potentially enables the distribution of propagated CEnCs to secondary sites for clinical use, as well as mitigating the time-sensitive aspect currently present with donor tissue. Previously described approaches generally used freezing solution containing 10% DMSO within a serum-containing medium for the cryo-preservation of CEnCs 19 , 40 . Despite being frequently used as a cryo-protectant, DMSO is also known to have multiple effects on cellular function, affecting cell cycle, as well as apoptosis in certain cell types 42 , and is a known cell-differentiating agent 43 .…”

Section: Discussionmentioning

confidence: 99%

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Regulatory Compliant Tissue-Engineered Human Corneal Endothelial Grafts Restore Corneal Function of Rabbits with Bullous Keratopathy

Peh

Ang

Lwin

et al. 2017

Sci Rep

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Corneal transplantation is the only treatment available to restore vision for individuals with blindness due to corneal endothelial dysfunction. However, severe shortage of available donor corneas remains a global challenge. Functional regulatory compliant tissue-engineered corneal endothelial graft substitute can alleviate this reliance on cadaveric corneal graft material. Here, isolated primary human corneal endothelial cells (CEnCs) propagated using a dual media approach refined towards regulatory compliance showed expression of markers indicative of the human corneal endothelium, and can be tissue-engineered onto thin corneal stromal carriers. Both cellular function and clinical adaptability was demonstrated in a pre-clinical rabbit model of bullous keratopathy using a tissue-engineered endothelial keratoplasty (TE-EK) approach, adapted from routine endothelial keratoplasty procedure for corneal transplantation in human patients. Cornea thickness of rabbits receiving TE-EK graft gradually reduced over the first two weeks, and completely recovered to a thickness of approximately 400 µm by the third week of transplantation, whereas corneas of control rabbits remained significantly thicker over 1,000 µm (p < 0.05) throughout the course of the study. This study showed convincing evidence of the adaptability of the propagated CEnCs and their functionality via a TE-EK approach, which holds great promises in translating the use of cultured CEnCs into the clinic.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Regulatory Compliant Tissue-Engineered Human Corneal Endothelial Grafts Restore Corneal Function of Rabbits with Bullous Keratopathy

Peh

Ang

Lwin

et al. 2017

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Moreover, FBS‐supplemented medium was used for the initiation of the primary culture, with HS‐culture starting only at Passage 1. In a more recent study from the same group (Vianna et al, ), CEC culture was initiated in both HS and FBS supplemented media, which were also used for cryopreservation upon addition of DMSO. Growth characteristics and morphology of HS‐ and FBS‐cultured cells after the freeze and thaw cycle were similar.…”

Section: Current Alternatives To the Use Of Fbs In Ec Culturementioning

confidence: 99%

Fetal bovine serum-free culture of endothelial progenitor cells-progress and challenges

Bauman

Granja

Barrias

2018

J Tissue Eng Regen Med

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Two decades after the first report on endothelial progenitor cells (EPC), their key role in postnatal vasculogenesis and vascular repair is well established. The therapeutic potential of EPC and their growing use in clinical trials calls for the development of more robust, reproducible, and safer methods for the in vitro expansion and maintenance of these cells. Despite many limitations associated with its usage, fetal bovine serum (FBS) is still widely applied as a cell culture supplement. Although different approaches aiming at establishing FBS-free culture have been developed for many cell types, adequate solutions for endothelial cells, and for EPC in particular, are still scarce, possibly due to the multiple challenges that have to be faced when culturing these cells. In this review, we provide a brief overview on the therapeutic relevance of EPC and critically analyse the available literature on FBS-free endothelial cell culture methods, including xeno-free, serum-free, and chemically defined systems.

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“…С возрастом часть клеток гибнет и целостность клеточного слоя сохраняется за счет оставшихся живых клеток, которые также формируют сплошной клеточный слой, но с меньшим количеством клеток. В связи этим до настоящего времени одним из основных критериев функциональности эндотелия и пригодности препарата роговицы для трансплантации является плотность клеток эндотелия (ECD), которая снижается с возрастом донора (Vianna et al, 2016). В различных глазных банках приняты минимальные пороговые значения ECD: от 2 500 до 2 000 (мм -2 ).…”

Section: Discussionunclassified

Study of the Effect of Hypothermic Conservation on the Intracellular Sodium Concentration in the Endothelium of Corneal Transplants

Baturina¹,

Palchikova²,

Konev³

et al. 2018

Vestn. VOGiS

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Endothelial keratoplasty has become the treatment of choice for corneal endothelial dysfunction. Advancements in the surgical treatment of corneal endothelial diseases depend on progress in graft conservation and its related advantages in assessing the suitability of grafts for transplantation. Transport of water and ions by cornea endothelium is important for the optic properties of cornea. In this work, we study the intracellular sodium concentration in cornea endothelial cells in samples of pig cornea that underwent hypothermic conservation for 1 and 10 days and endothelial cells of human cornea grafts after 10-day conservation. The concentration of intracellular sodium in preparations of endothelial cells was assayed using fluorescent dye SodiumGreen. The fluorescent images were analyzed with the custom-made computer program CytoDynamics. An increased level of intracellular sodium was shown in the endothelium after 10-day conservation in comparison with one-day conservation (pig samples). Sodium permeability of pig endothelial cell plasma membranes significantly decreased in these samples. Assessment of intracellular sodium in human cornea endothelium showed a higher level – as was in analogues pig samples of the corneal endothelium. The assay of the intracellular sodium balance concentration established in endothelial cells after hypothermic conservation in mediums L-15 and Optisol-GS showed a significant advantage of specialized me dium Optisol-GS. The balanced intracellular concentration after 10 days of hypothermic conservation was significantly lower in cells incubated at 4 °C in Optisol-GS (L-15, 128 ± 14, n = 15; Optisol-GS, 108 ± 14, n = 11; mM, p < 0.001). Intracellular sodium concentration could be a useful parameter for assessing cornea endothelium cell viability.

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Characterization of cryopreserved primary human corneal endothelial cells cultured in human serum-supplemented media

Cited by 8 publications

References 16 publications

Regulatory Compliant Tissue-Engineered Human Corneal Endothelial Grafts Restore Corneal Function of Rabbits with Bullous Keratopathy

Regulatory Compliant Tissue-Engineered Human Corneal Endothelial Grafts Restore Corneal Function of Rabbits with Bullous Keratopathy

Fetal bovine serum-free culture of endothelial progenitor cells-progress and challenges

Study of the Effect of Hypothermic Conservation on the Intracellular Sodium Concentration in the Endothelium of Corneal Transplants

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