Enhancing the Structural Maturation and Attachment of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes on Textile Surfaces
Mansikkala, Leo Tuomas (2017)
2017
Biotekniikka
Teknis-luonnontieteellinen tiedekunta - Faculty of Natural Sciences
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Hyväksymispäivämäärä
2017-03-08
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-201703131159
https://urn.fi/URN:NBN:fi:tty-201703131159
Tiivistelmä
Cardiovascular diseases are one of the leading causes of death in the world. In 2012 ~30% of all deaths were due to cardiovascular diseases. There is a need for new research, drug testing and treatment methods. Tissue engineering and cell culturing can be used to create new models and in future even treatments. Human cardiomyocytes (CMs) have been hard to obtain for research, requiring heart biopsies or human embryonic stem cells. That has been changed by the discovery of human induced pluripotent stem cells (hiPSC). Cells harvested from adults can be induced back to pluripotent stem cells that can be then differentiated to hiPSC derived CMs (hiPSC-CMs). HiPSC maintain the genome of the patient, which allows the study of specific changes and their effects in the genome. One problem with hiPSC-CMs is that they do not have all the functional and morphological properties of adult CMs when cultured on normal in vitro conditions. The aim of this study was to determine if using differently coated textiles as culturing surfaces, would create structurally more mature hiPSC-CM.
The hiPSC were differentiated into CMs by END-2 and small molecule method. The produced hiPSC-CM were cultured on textiles coated with either gelatin, collagen, Matrigel or Geltrex. To increase the attachment of gelatin, some of the textiles samples were modified with dopamine or plasma treatment. The controls were grown on gelatin and Geltrex coated cover slips. The analysis was made with immunocytochemistry and fluorescence microscopy. The samples were labelled with antibodies against sarcomere proteins. Live/dead staining was used to determine cell attachment. The shape of hiPSC-CM and its sarcomere organization, orientation and length were analyzed from the immunostaining images using Cytospectre software.
The structural maturation of hiPSC-CMs was evaluated between all coatings and textile structures and they were compared to the hiPSC-CMs grown on coverslips. The cells attached to the textiles, but large portion of the cells passed through the textiles and was left on the bottom of the growth well. The textile grown hiPSC-CMs had more adult like shape and sarcomere structure, but there was no difference in sarcomere lengths. No clear difference between coatings was detected.
Based on the result, the textile structure has a bigger effect on structural maturity than coating material, which only seem to the affect attachment of the cells. This study only established the increase in structural maturity. In the future, it has to be tested if these changes cause any functional improvements.
The hiPSC were differentiated into CMs by END-2 and small molecule method. The produced hiPSC-CM were cultured on textiles coated with either gelatin, collagen, Matrigel or Geltrex. To increase the attachment of gelatin, some of the textiles samples were modified with dopamine or plasma treatment. The controls were grown on gelatin and Geltrex coated cover slips. The analysis was made with immunocytochemistry and fluorescence microscopy. The samples were labelled with antibodies against sarcomere proteins. Live/dead staining was used to determine cell attachment. The shape of hiPSC-CM and its sarcomere organization, orientation and length were analyzed from the immunostaining images using Cytospectre software.
The structural maturation of hiPSC-CMs was evaluated between all coatings and textile structures and they were compared to the hiPSC-CMs grown on coverslips. The cells attached to the textiles, but large portion of the cells passed through the textiles and was left on the bottom of the growth well. The textile grown hiPSC-CMs had more adult like shape and sarcomere structure, but there was no difference in sarcomere lengths. No clear difference between coatings was detected.
Based on the result, the textile structure has a bigger effect on structural maturity than coating material, which only seem to the affect attachment of the cells. This study only established the increase in structural maturity. In the future, it has to be tested if these changes cause any functional improvements.