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Optimization of polycaprolactone - based nanofiber matrices for the cultivation of corneal endothelial cells.

ABSTRACT: Posterior lamellar transplantation of the eye' s cornea (DSAEK, DMEK) currently is the gold standard for treating patients with corneal endothelial cell and back surface pathologies resulting in functional impairment. An artificial biomimetic graft carrying human corneal endothelium could minimize the dependency on human donor corneas giving access to this vision-restoring surgery to large numbers of patients, thus reducing current long waiting lists. In this study, four groups of electrospun nanofibrous scaffolds were compared: polycaprolactone (PCL), PCL/collagen, PCL/gelatin and PCL/chitosan. Each of the scaffolds were tissue-engineered with human corneal endothelial cells (HCEC-B4G12) and analyzed with regard to their potential application as artificial posterior lamellar grafts. Staining with ZO-1 and Na⁺/K⁺-ATPase antibodies revealed intact cell functionalities. It could be shown, that blending leads to decreasing contact angle, whereby a heterogeneous blend morphology could be revealed. Scaffold cytocompatibility could be confirmed for all groups via live/dead staining, whereby a significant higher cell viability could be observed for the collagen and gelatine blended matrices with 97 ± 3% and 98 ± 2% living cells respectively. TEM images show the superficial anchoring of the HCECs onto the scaffolds. This work emphasizes the benefit of blended PCL nanofibrous scaffolds for corneal endothelial keratoplasty.

SUBMITTER: Himmler M

PROVIDER: S-EPMC8458296 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundGlobal shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology.ResultsEstablished primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology.ConclusionsOur results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

Project description:BACKGROUND:Induced pluripotent stem-cell derived endothelial cells (iPSC-ECs) can be generated from any somatic cell and their iPSC sources possess unlimited self-renewal. Previous demonstration of their proangiogenic activity makes them a promising cell type for treatment of ischemic injury. As with many other stem cell approaches, the low rate of in-vivo survival has been a major limitation to the efficacy of iPSC-ECs to date. In this study, we aimed to increase the in-vivo lifetime of iPSC-ECs by culturing them on electrospun polycaprolactone (PCL)/gelatin scaffolds, before quantifying the subsequent impact on their proangiogenic function. METHODS:iPSC-ECs were isolated and stably transfected with a luciferase reporter to facilitate quantification of cell numbers and non-invasive imaging in-vivo PCL/gelatin scaffolds were engineered using electrospinning to obtain woven meshes of nanofibers. iPSC-ECs were cultured on scaffolds for 7 days. Subsequently, cell growth and function were assessed in vitro followed by implantation in a mouseback subcutaneous model for 7 days. RESULTS:Using a matrix of conditions, we found that scaffold blends with ratios of PCL:gelatin of 70:30 (PG73) spun at high flow rates supported the greatest levels of iPSC-EC growth, retention of phenotype, and function in vitro. Implanting iPSC-ECs seeded on PG73 scaffolds in vivo improved their survival up to 3 days, compared to cells directly injected into control wounds, which were no longer observable within 1 h. Enhanced engraftment improved blood perfusion, observed through non-invasive laser Doppler imaging. Immunohistochemistry revealed a corresponding increase in host angiogenic mechanisms characterized by the enhanced recruitment of macrophages and the elevated expression of proangiogenic cytokines vascular endothelial growth factor and placental growth factor. CONCLUSIONS:Knowledge of these mechanisms combined with a deeper understanding of the scaffold parameters influencing this function provides the groundwork for optimizing future iPSC-EC therapies utilizing engraftment platforms. The development of combined scaffold and iPSC-EC therapies could ultimately improve therapeutic angiogenesis and the treatment of ischemic injury.

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Optimization of polycaprolactone - based nanofiber matrices for the cultivation of corneal endothelial cells.

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