Limits...
Osteogenic differentiation of mesenchymal stromal cells in two-dimensional and three-dimensional cultures without animal serum.

Castrén E, Sillat T, Oja S, Noro A, Laitinen A, Konttinen YT, Lehenkari P, Hukkanen M, Korhonen M - Stem Cell Res Ther (2015)

Bottom Line: Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks.In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment.As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Anatomy, Biomedicum Helsinki, University of Helsinki, PO Box 63, Helsinki, Finland. eeva.castren@helsinki.fi.

ABSTRACT

Introduction: Bone marrow-derived mesenchymal stromal cells (MSCs) have been intensely studied for the purpose of developing solutions for clinical tissue engineering. Autologous MSCs can potentially be used to replace tissue defects, but the procedure also carries risks such as immunization and xenogeneic infection. Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks. Altered media could, however, influence stem cell differentiation and we address this experimentally.

Methods: We examined human MSC differentiation into the osteoblast lineage using in vitro two- and three-dimensional cultures with PLP or FCS as cell culture medium supplements. Differentiation was followed by quantitative polymerase chain reaction, and alkaline phosphatase activity, matrix formation and matrix calcium content were quantified.

Results: Three-dimensional culture, where human MSCs were grown on collagen sponges, markedly stimulated osteoblast differentiation; a fourfold increase in calcium deposition could be observed in both PLP and FCS groups. PLP-grown cells showed robust osteogenic differentiation both in two- and three-dimensional MSC cultures. The calcium content of the matrix in the two-dimensional PLP group at day 14 was 2.2-fold higher in comparison to the FCS group (p < 0.0001), and at day 21 it was still 1.3-fold higher (p < 0.001), suggesting earlier calcium accumulation to the matrix in the PLP group. This was supported by stronger Alizarin Red staining in the PLP group at day 14. In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment. In three-dimensional experiments, the PLP and FCS groups behaved more congruently, except for the alkaline phosphatase activity and mRNA levels which were markedly increased by PLP.

Conclusions: Human PLP was at least equal to FCS in supporting osteogenic differentiation of human MSCs in two- and three-dimensional conditions; however, proliferation was inferior. As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

No MeSH data available.


Related in: MedlinePlus

Detection of mineralized matrix and calcium deposition in two-dimensional (2D) cell culture. Cells were cultured in 2D osteogenic conditions up to 21 days in FCS and PLP culture media. On days a 14 and b 21, the cell cultures were fixed and stained with Alizarin Red, after which they were dried and photographed. c Thereafter, they were turned into binary images and optical densities were measured. d Deposited calcium was dissolved in HCl and quantified on days 7, 14 and 21 of culture using an o-cresolphthalein complexone reagent using a standard curve as reference. CTRL control, DIFF differentiation, FCS fetal calf serum, PLP platelet lysate and plasma
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4562352&req=5

Fig4: Detection of mineralized matrix and calcium deposition in two-dimensional (2D) cell culture. Cells were cultured in 2D osteogenic conditions up to 21 days in FCS and PLP culture media. On days a 14 and b 21, the cell cultures were fixed and stained with Alizarin Red, after which they were dried and photographed. c Thereafter, they were turned into binary images and optical densities were measured. d Deposited calcium was dissolved in HCl and quantified on days 7, 14 and 21 of culture using an o-cresolphthalein complexone reagent using a standard curve as reference. CTRL control, DIFF differentiation, FCS fetal calf serum, PLP platelet lysate and plasma

Mentions: For detection of deposited mineralized matrix in the differentiating MSC cultures, the cultures were stained with Alizarin Red. By day 14 of differentiation, the PLP-grown cells displayed more intensive Alizarin Red staining than the FCS cultures (p < 0.01; Fig. 4a and c). By day 21, both PLP- and FCS-grown cells showed evidence of similarly rich mineralization (Fig. 4b and c). Quantitative data, acquired by measuring OD values, confirmed the visual impression (Fig. 4c).Fig. 4


Osteogenic differentiation of mesenchymal stromal cells in two-dimensional and three-dimensional cultures without animal serum.

Castrén E, Sillat T, Oja S, Noro A, Laitinen A, Konttinen YT, Lehenkari P, Hukkanen M, Korhonen M - Stem Cell Res Ther (2015)

Detection of mineralized matrix and calcium deposition in two-dimensional (2D) cell culture. Cells were cultured in 2D osteogenic conditions up to 21 days in FCS and PLP culture media. On days a 14 and b 21, the cell cultures were fixed and stained with Alizarin Red, after which they were dried and photographed. c Thereafter, they were turned into binary images and optical densities were measured. d Deposited calcium was dissolved in HCl and quantified on days 7, 14 and 21 of culture using an o-cresolphthalein complexone reagent using a standard curve as reference. CTRL control, DIFF differentiation, FCS fetal calf serum, PLP platelet lysate and plasma
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4562352&req=5

Fig4: Detection of mineralized matrix and calcium deposition in two-dimensional (2D) cell culture. Cells were cultured in 2D osteogenic conditions up to 21 days in FCS and PLP culture media. On days a 14 and b 21, the cell cultures were fixed and stained with Alizarin Red, after which they were dried and photographed. c Thereafter, they were turned into binary images and optical densities were measured. d Deposited calcium was dissolved in HCl and quantified on days 7, 14 and 21 of culture using an o-cresolphthalein complexone reagent using a standard curve as reference. CTRL control, DIFF differentiation, FCS fetal calf serum, PLP platelet lysate and plasma
Mentions: For detection of deposited mineralized matrix in the differentiating MSC cultures, the cultures were stained with Alizarin Red. By day 14 of differentiation, the PLP-grown cells displayed more intensive Alizarin Red staining than the FCS cultures (p < 0.01; Fig. 4a and c). By day 21, both PLP- and FCS-grown cells showed evidence of similarly rich mineralization (Fig. 4b and c). Quantitative data, acquired by measuring OD values, confirmed the visual impression (Fig. 4c).Fig. 4

Bottom Line: Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks.In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment.As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine, Anatomy, Biomedicum Helsinki, University of Helsinki, PO Box 63, Helsinki, Finland. eeva.castren@helsinki.fi.

ABSTRACT

Introduction: Bone marrow-derived mesenchymal stromal cells (MSCs) have been intensely studied for the purpose of developing solutions for clinical tissue engineering. Autologous MSCs can potentially be used to replace tissue defects, but the procedure also carries risks such as immunization and xenogeneic infection. Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks. Altered media could, however, influence stem cell differentiation and we address this experimentally.

Methods: We examined human MSC differentiation into the osteoblast lineage using in vitro two- and three-dimensional cultures with PLP or FCS as cell culture medium supplements. Differentiation was followed by quantitative polymerase chain reaction, and alkaline phosphatase activity, matrix formation and matrix calcium content were quantified.

Results: Three-dimensional culture, where human MSCs were grown on collagen sponges, markedly stimulated osteoblast differentiation; a fourfold increase in calcium deposition could be observed in both PLP and FCS groups. PLP-grown cells showed robust osteogenic differentiation both in two- and three-dimensional MSC cultures. The calcium content of the matrix in the two-dimensional PLP group at day 14 was 2.2-fold higher in comparison to the FCS group (p < 0.0001), and at day 21 it was still 1.3-fold higher (p < 0.001), suggesting earlier calcium accumulation to the matrix in the PLP group. This was supported by stronger Alizarin Red staining in the PLP group at day 14. In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment. In three-dimensional experiments, the PLP and FCS groups behaved more congruently, except for the alkaline phosphatase activity and mRNA levels which were markedly increased by PLP.

Conclusions: Human PLP was at least equal to FCS in supporting osteogenic differentiation of human MSCs in two- and three-dimensional conditions; however, proliferation was inferior. As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

No MeSH data available.


Related in: MedlinePlus