Analysis of Attachment, Proliferation and Maturation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells on Specific Substrata
Kurkela, Olli (2011)
2011
Biotekniikan koulutusohjelma
Luonnontieteiden ja ympäristötekniikan tiedekunta - Faculty of Science and Environmental Engineering
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Hyväksymispäivämäärä
2011-06-08
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-2011062214741
https://urn.fi/URN:NBN:fi:tty-2011062214741
Tiivistelmä
Most severe degenerative diseases of retina are often due to malfunctions of retinal pigment epithelium (RPE). Absence of effective treatments has led to development of cell-biomaterial constructs with the aim of creating RPE equivalents for transplantation. Presently, the poor biocompatibility of allologous and xenologous culture substrata in addition with limited amount of source tissue poses the major issues. Well-defined synthetic substrata together with utilization of human embryonic stem cell-derived RPE cells (hESC RPE) are suggested to be potential solutions. In addition, need exists for an effective method to determine the developmental status of cells during the culturing period. This need could be addressed with automated image analysis.
The aim of this thesis was to examine the capability of a few specific cell culture substrata to enable attachment, proliferation and maturation of hESC RPE cells. Study included total of 17 xeno-free synthetic materials including 12 BioMaDe Gelators, Purecoat amine and carboxyl, poly(D,L-lactic-co-glycolic acid) (75:25), poly(D,L-lactic acid) (96:4) and poly(L-lactic acid-co-?-caprolactone) (70:30). In addition five materials with natural-origin were studied including chitosan, type I collagen, Matrigel and Substrate X. Type IV collagen was used as control. Growth and maturation were monitored by taking images with specific time intervals. At the end point cellular developmental status was determined by assessing the expression of maturation specific mRNAs by PCR techniques and proteins by immunofluorescence microscopy. In addition, images were used to determine the potential of ImageJ-software as user-friendly image analysis tool for RPE cell analysis.
Study demonstrated poor attachment and cell survival on every xeno-free synthetic substrate with cells retaining their initial developmental phase throughout the culturing period, which was supported by gene expression analysis. On the contrary, cells on natural materials attached and proliferated readily. Maturity was further confirmed with immunofluorescence labeling. Image analysis with ImageJ, in turn, confronted many problems mainly arising from heterogeneity of the images.
As a conclusion, xeno-free synthetic materials tested in this study show low potential as RPE cell substrata. However, means to enhance their performance are suggested. Despite the good results obtained with natural materials, their ill-defined structure prone to alterations in physiological conditions remains an obstacle for entering clinical experiments. Further experiments should concentrate on combining the strengths of both approaches, that is, incorporation of attachment-related functional groups into well-defined xeno-free synthetic body. In order to increase image homogeneity imaging conditions should be more carefully considered. This way the benefits of automated image analysis could be more effectively exploited. /Kir11
The aim of this thesis was to examine the capability of a few specific cell culture substrata to enable attachment, proliferation and maturation of hESC RPE cells. Study included total of 17 xeno-free synthetic materials including 12 BioMaDe Gelators, Purecoat amine and carboxyl, poly(D,L-lactic-co-glycolic acid) (75:25), poly(D,L-lactic acid) (96:4) and poly(L-lactic acid-co-?-caprolactone) (70:30). In addition five materials with natural-origin were studied including chitosan, type I collagen, Matrigel and Substrate X. Type IV collagen was used as control. Growth and maturation were monitored by taking images with specific time intervals. At the end point cellular developmental status was determined by assessing the expression of maturation specific mRNAs by PCR techniques and proteins by immunofluorescence microscopy. In addition, images were used to determine the potential of ImageJ-software as user-friendly image analysis tool for RPE cell analysis.
Study demonstrated poor attachment and cell survival on every xeno-free synthetic substrate with cells retaining their initial developmental phase throughout the culturing period, which was supported by gene expression analysis. On the contrary, cells on natural materials attached and proliferated readily. Maturity was further confirmed with immunofluorescence labeling. Image analysis with ImageJ, in turn, confronted many problems mainly arising from heterogeneity of the images.
As a conclusion, xeno-free synthetic materials tested in this study show low potential as RPE cell substrata. However, means to enhance their performance are suggested. Despite the good results obtained with natural materials, their ill-defined structure prone to alterations in physiological conditions remains an obstacle for entering clinical experiments. Further experiments should concentrate on combining the strengths of both approaches, that is, incorporation of attachment-related functional groups into well-defined xeno-free synthetic body. In order to increase image homogeneity imaging conditions should be more carefully considered. This way the benefits of automated image analysis could be more effectively exploited. /Kir11