OBJECTIVE: Patients of corneal opacity are increasing year by year. The treatment for this disease is relatively straightforward: corneal transplant from a human donor. However, there is a global shortage of donor corneas, leading to many patients not being able to receive the treatment even though the procedure itself is well developed. Scientists have continuously tried to find alternative methods of human corneal transplant. Recently, the porcine cornea has become a research focus as a transplantation alternative because of mechanical and morphological similarities to the human cornea. This study focused on analyzing the porcine corneal endothelial cell (PCEC), specifically its density-dependent characteristics in vitro and its proliferation capabilities. The hypothesis was tested that PCECs could proliferate most efficiently under a high-density seeding environment and could maintain cell efficacy for 5 passages.
METHODS: PCECs were isolated from three fresh porcine corneas and seeded into 24-well petri dishes at 10,000 cells/cm2 (high density) and 6,700 cells/cm2 (low density). Cells were cultured for 96 hours before subculturing with the same conditions onto the next passage. Samples from doubling times of each passage were collected and analyzed, and tight junction markers were stained with proteins ZO-1 (zonula occludens-1) and N-cadherin to test for tight junction functionality.
RESULTS: The high-density group averaged a faster doubling time at 38.3 hours (n = 8) compared with the doubling time of the low-density group at 43.2 hours (n = 8). PCECs were able to maintain proliferation and matured for 8 passages until losing cell morphology (more drastic for the high-density group) and cell tight junction (both groups) at the ninth passage. Doubling times were also increased drastically for both density groups at the ninth passage.
CONCLUSION: Examination of PCEC growing conditions led to the discovery that growth density is crucial to the overall quality of the corneal endothelial cells. From these data and past research, high-density growth conditions of ≥10,000 cells/cm2 were more beneficial than a low-density environment of 6,700 cells/cm2. PCECs were able to maintain tight junction functionality and cell morphology for 8 passages when grown to subconfluency. Based on the analysis of these results, the PCEC appears to be a more accessible corneal study target because of the many characteristics similar to the human corneal endothelial cell (HCEC), making it a possible alternative in medical transplant research in the future. This potential of the PCEC, if successfully developed, will increase the arsenal of choices for doctors and researchers around the world in the combat against corneal opacity.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/45539 |
| Date | 30 January 2023 |
| Creators | Chang, Howard |
| Contributors | Spencer, Jean L. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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