Dynamic culture of human adipose stem cells in a flow perfusion bioreactor
Vuornos, Kaisa (2016)
2016
Materiaalitekniikan koulutusohjelma
Teknisten tieteiden tiedekunta - Faculty of Engineering Sciences
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Hyväksymispäivämäärä
2016-12-07
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-201611244768
https://urn.fi/URN:NBN:fi:tty-201611244768
Tiivistelmä
Regenerative medicine aims to restore or replace damaged tissue functions. Tissue engineering offers a solution to the growing shortage of suitable tissue and organ donors by combining stem cells with biomaterials and soluble factors. Bone defects and acute traumas together with increased life expectancy augment the demand for new tissue engineered bone tissue constructs.
Among multipotent mesenchymal stem cells, human adipose stem cells (hASCs) are an abundant and accessible source of adult stem cells with capacity to proliferate and differentiate in vitro towards at least fat, bone, muscle, cartilage, and tendon tissues under appropriate conditions. With autologous cells, the risk of adverse immunological reactions is reduced.
The aim of this work was to test the suitability of a new flow perfusion bioreactor for aseptic cell culture. The dynamic fluid flow was used to induce osteogenic differentiation of the hASCs in novel supercritical CO2 processed polymer composite scaffolds of polylactide-co-poly-ε-caprolactone with 40 wt-% β-tricalcium phosphate granules (PLCL--TCP).
Biochemical analysis methods well established in the field were used. Flow cytometric cluster of differentiation marker expression analysis was used to verify the stem cell properties of the hASCs. Cell viability and adhesion was qualitatively analyzed with Live/Dead fluorescence staining. Cell number was analyzed using a quantitative assay based on the total amount of DNA in the sample. The hASC osteogenic differentiation was assessed by evaluating quantitative alkaline phosphatase (qALP) activity, total collagen content, and mineralization.
Cells were viable in all the conditions. Higher hASC proliferation was obtained with the perfusion flow bioreactor in both the flow rate and structure comparison experiments and uniform cell distribution was gained for the channel scaffolds under perfusion. In the dynamic condition, the results for the qALP, total collagen content, and mineralization analyses were similar or lower compared to the static control in all the experiments. No osteogenic differentiation of the hASCs was achieved in the flow perfusion bioreactor with basic maintenance cell culture medium without added chemical factors.
Further experiments are needed to define the functional cell culture conditions and fluid flow parameters in the bioreactor to support hASC osteogenic differentiation. The new bioreactor system is suitable for aseptic cell culture, easy to use and cost-effective.
Among multipotent mesenchymal stem cells, human adipose stem cells (hASCs) are an abundant and accessible source of adult stem cells with capacity to proliferate and differentiate in vitro towards at least fat, bone, muscle, cartilage, and tendon tissues under appropriate conditions. With autologous cells, the risk of adverse immunological reactions is reduced.
The aim of this work was to test the suitability of a new flow perfusion bioreactor for aseptic cell culture. The dynamic fluid flow was used to induce osteogenic differentiation of the hASCs in novel supercritical CO2 processed polymer composite scaffolds of polylactide-co-poly-ε-caprolactone with 40 wt-% β-tricalcium phosphate granules (PLCL--TCP).
Biochemical analysis methods well established in the field were used. Flow cytometric cluster of differentiation marker expression analysis was used to verify the stem cell properties of the hASCs. Cell viability and adhesion was qualitatively analyzed with Live/Dead fluorescence staining. Cell number was analyzed using a quantitative assay based on the total amount of DNA in the sample. The hASC osteogenic differentiation was assessed by evaluating quantitative alkaline phosphatase (qALP) activity, total collagen content, and mineralization.
Cells were viable in all the conditions. Higher hASC proliferation was obtained with the perfusion flow bioreactor in both the flow rate and structure comparison experiments and uniform cell distribution was gained for the channel scaffolds under perfusion. In the dynamic condition, the results for the qALP, total collagen content, and mineralization analyses were similar or lower compared to the static control in all the experiments. No osteogenic differentiation of the hASCs was achieved in the flow perfusion bioreactor with basic maintenance cell culture medium without added chemical factors.
Further experiments are needed to define the functional cell culture conditions and fluid flow parameters in the bioreactor to support hASC osteogenic differentiation. The new bioreactor system is suitable for aseptic cell culture, easy to use and cost-effective.