The effects of incubation of porcine RPE cells
Laitinen, Tomi (2021)
2021
Lääketieteen lisensiaatin tutkinto-ohjelma - Licentiate's Programme in Medicine
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2021-11-29
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202111228558
https://urn.fi/URN:NBN:fi:tuni-202111228558
Tiivistelmä
Introduction
Retinal pigment epithelium is a monolayer of cells between the choroid and neural retina. It is involved in pathogenesis of age-related macular degeneration (AMD) which currently has no curative treatment. Our studies have focused on differentiated RPE cells from human embryonic stem cells (hESCs) which have incomplete maturation in in vitro conditions. Due to this, we wanted to study native RPE cells. Aim of the study was to optimize a preparation method for the RPE cell layer and after that, to induce cellular stress on RPE cells.
Methods
Porcine eyes were gathered from a slaughterhouse. The eyes were prepared and labeled with immunohistochemistry against specific proteins. Samples were incubated for 0h, 3h or 24h before labeling. Immunolabeled samples were imaged with fluorescence microscope.
Results
Samples without incubation showed several patches of intact RPE cells. After 3 hours of incubation, samples still had an intact RPE cell layer but certain differences could be observed between non-incubated samples and samples that were incubated for 3 hours. After 24 hours, no RPE cell layer could be detected in the samples.
Discussion
The preparation method was successful in keeping the RPE cell layer intact. The cells potentially could survive 3 hours of incubation but not 24 hours of incubation. Further studies can be conducted by comparing RPE characteristics between native cells with RPE cells derived from hESCs using this preparation method.
Retinal pigment epithelium is a monolayer of cells between the choroid and neural retina. It is involved in pathogenesis of age-related macular degeneration (AMD) which currently has no curative treatment. Our studies have focused on differentiated RPE cells from human embryonic stem cells (hESCs) which have incomplete maturation in in vitro conditions. Due to this, we wanted to study native RPE cells. Aim of the study was to optimize a preparation method for the RPE cell layer and after that, to induce cellular stress on RPE cells.
Methods
Porcine eyes were gathered from a slaughterhouse. The eyes were prepared and labeled with immunohistochemistry against specific proteins. Samples were incubated for 0h, 3h or 24h before labeling. Immunolabeled samples were imaged with fluorescence microscope.
Results
Samples without incubation showed several patches of intact RPE cells. After 3 hours of incubation, samples still had an intact RPE cell layer but certain differences could be observed between non-incubated samples and samples that were incubated for 3 hours. After 24 hours, no RPE cell layer could be detected in the samples.
Discussion
The preparation method was successful in keeping the RPE cell layer intact. The cells potentially could survive 3 hours of incubation but not 24 hours of incubation. Further studies can be conducted by comparing RPE characteristics between native cells with RPE cells derived from hESCs using this preparation method.