Optimizing xeno-free culture conditions for human embryonic stem cells to correspond GMP-quality standards
KETOLA, SARITA (2006)
2006
Biokemia - Biochemistry
Lääketieteellinen tiedekunta - Faculty of Medicine
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Hyväksymispäivämäärä
2006-05-08
Julkaisun pysyvä osoite on
https://urn.fi/urn:nbn:fi:uta-1-15638
https://urn.fi/urn:nbn:fi:uta-1-15638
Tiivistelmä
Human embryonic stem cells (hESCs) can offer an unlimited cell source for cellular transplantation, but the currently used culture conditions contain animal-derived products that bear a risk of transmitting animal pathogens and causing a rejection when cells
are transplanted. In addition, the laborious production of feeders needed for hESC culture and the difficult mechanical passaging of hESCs make the large-scale production of hESCs difficult. The first aim of this study was to optimize culture conditions for hESCs towards animal-free conditions by testing totally animal-free serum replacements (SR-3 and X-Vivo 20) and basic fibroblast growth factor (bFGF). Other aims of the study were to find an easier way for hESC passaging and to test the possibility of growing hESCs without feeder cells using biomaterials.
The differentiation stage of the hESC colonies was determined during the testing by morphology and later confirmed with immunochemistry using markers specific for undifferentiated hESCs (AP, Oct-4 and Nanog) and for differentiated hESCs (SSEA-1).
The hESCs grown with the SR-3 and X-Vivo 20 were all differentiated where as some of the hESCs grown with the test bFGF were undifferentiated. However, the amount of these cells was very low. The hESCs passaged with animal-free enzyme Tryple were mostly undifferentiated and the amount of these cells was similar with control. In addition, the cells had normal 46,XX karyotype after 31 passages. The embryoid bodies (EBs) differentiated from these hESCs expressed genes and proteins common to the three embryonic germ layers based on the reverse transcriptase-PCR and immunochemistry analysis, showing the pluripotency of the cells. The tested biomaterials allowed the attachment of hESCs in the presence of conditioned medium, but the undifferentiated growth of hESCs could not be determined. However, the cells plated back on feeders after one passage on biomaterials, were mostly differentiated.
The tested SR-3, X-Vivo 20 and bFGF as well as the biomaterials were not able to maintain the undifferentiated growth of hESCs better than the control conditions, but the Tryple passaging method supported the growth of hESCs well. Although it was not faster way than mechanical passaging, it makes the passaging of thin hESC colonies easier and the colonies could be split into smaller pieces than with mechanical passaging, resulting in faster expanded growth of hESCs.
Asiasanat: human embryonic stem cell, GMP, serum replacement, bFGF, enzymatic passaging, biomaterials
are transplanted. In addition, the laborious production of feeders needed for hESC culture and the difficult mechanical passaging of hESCs make the large-scale production of hESCs difficult. The first aim of this study was to optimize culture conditions for hESCs towards animal-free conditions by testing totally animal-free serum replacements (SR-3 and X-Vivo 20) and basic fibroblast growth factor (bFGF). Other aims of the study were to find an easier way for hESC passaging and to test the possibility of growing hESCs without feeder cells using biomaterials.
The differentiation stage of the hESC colonies was determined during the testing by morphology and later confirmed with immunochemistry using markers specific for undifferentiated hESCs (AP, Oct-4 and Nanog) and for differentiated hESCs (SSEA-1).
The hESCs grown with the SR-3 and X-Vivo 20 were all differentiated where as some of the hESCs grown with the test bFGF were undifferentiated. However, the amount of these cells was very low. The hESCs passaged with animal-free enzyme Tryple were mostly undifferentiated and the amount of these cells was similar with control. In addition, the cells had normal 46,XX karyotype after 31 passages. The embryoid bodies (EBs) differentiated from these hESCs expressed genes and proteins common to the three embryonic germ layers based on the reverse transcriptase-PCR and immunochemistry analysis, showing the pluripotency of the cells. The tested biomaterials allowed the attachment of hESCs in the presence of conditioned medium, but the undifferentiated growth of hESCs could not be determined. However, the cells plated back on feeders after one passage on biomaterials, were mostly differentiated.
The tested SR-3, X-Vivo 20 and bFGF as well as the biomaterials were not able to maintain the undifferentiated growth of hESCs better than the control conditions, but the Tryple passaging method supported the growth of hESCs well. Although it was not faster way than mechanical passaging, it makes the passaging of thin hESC colonies easier and the colonies could be split into smaller pieces than with mechanical passaging, resulting in faster expanded growth of hESCs.
Asiasanat: human embryonic stem cell, GMP, serum replacement, bFGF, enzymatic passaging, biomaterials