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Effects of different sera conditions on olfactory ensheathing cells in vitro.

Lu M, Dong J, Lu T, Lv H, Yang P, Cheng Z, Li J, Liang B, Xu J, Li H, He X - Int J Mol Sci (2014)

Bottom Line: Compared with FBS, RS reduced OEC proliferation in relation to OEC counts (χ2 = 166.279, df = 1, p < 0.01), the optical density (OD) value in the MTT assay (χ2 = 34.730, df = 1, p < 0.01), and NT-3 concentration in the supernatant (χ2 = 242.997, df = 1, p < 0.01).There was statistically significant difference in NT-3 gene expression among different groups when the serum concentration was 15% (χ2 = 64.347, df = 1, p < 0.01).In conclusion, different serum conditions may be responsible for the variations in OEC proliferation and function.

View Article: PubMed Central - PubMed

Affiliation: Second Department of Orthopedics, Second Affiliated Hospital of Xi'an Jiaotong University, 157 West Five Road, Xincheng District, Xi'an 710004, Shaanxi, China. lumeng6020@163.com.

ABSTRACT
Transplantation of olfactory ensheathing cells (OEC) is a promising therapy in spinal cord injury (SCI) treatment. However, the therapeutic efficacy of this method is unstable due to unknown reasons. Considering the alterations in the culture environment that occur during OEC preparation for transplantation, we hypothesize that these changes may cause variations in the curative effects of this method. In this study, we compared OEC cultured in medium containing different types and concentrations of serum. After purification and passage, the OEC were cultured for 7 days in different media containing 5%, 10%, 15% or 20% fetal bovine serum (FBS) or rat serum (RS), or the cells were cultured in FBS-containing medium first, followed by medium containing RS. In another group, the OEC were first cultured in 10% FBS for 3 days and then cultured with rat spinal cord explants with 10% RS for another 4 days. An MTT assay and P75 neurotrophin receptor immunofluorescence staining were used to examine cell viability and OEC numbers, respectively. The concentration of neurotrophin-3 (NT-3), which is secreted by OEC into the culture supernatant, was detected using the enzyme-linked immunosorbent assay (ELISA). RT-PCR was applied to investigate the NT-3 gene expression in OEC according to different groups. Compared with FBS, RS reduced OEC proliferation in relation to OEC counts (χ2 = 166.279, df = 1, p < 0.01), the optical density (OD) value in the MTT assay (χ2 = 34.730, df = 1, p < 0.01), and NT-3 concentration in the supernatant (χ2 = 242.997, df = 1, p < 0.01). OEC cultured with spinal cord explants secreted less NT-3 than OEC cultured alone (F = 9.611, df = 5.139, p < 0.01). Meanwhile, the order of application of different sera was not influential. There was statistically significant difference in NT-3 gene expression among different groups when the serum concentration was 15% (χ2 = 64.347, df = 1, p < 0.01). In conclusion, different serum conditions may be responsible for the variations in OEC proliferation and function.

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Immunofluorescence staining of OEC cultured in DF-12 medium containing rat serum (RS) (concentrations: 5%, 10%, 15%, 20%) for 7 days. The concentrations of RS are listed on the left. OEC nuclei stained with DAPI are presented in the left column, P75 of OEC stained with Cy3 are presented in the middle column, and the right column is the merged nuclei and P75. Scale bar is 200 μm.
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ijms-16-00420-f002: Immunofluorescence staining of OEC cultured in DF-12 medium containing rat serum (RS) (concentrations: 5%, 10%, 15%, 20%) for 7 days. The concentrations of RS are listed on the left. OEC nuclei stained with DAPI are presented in the left column, P75 of OEC stained with Cy3 are presented in the middle column, and the right column is the merged nuclei and P75. Scale bar is 200 μm.

Mentions: After P75 immunofluorescence staining, an increased number of cells in the visual field were observed (Figure 1, Figure 2, Figure 3 and Figure 4). The immunofluorescence of OEC from Group E was shown in Figure 5. The number of OEC per unit area was shown in Figure 6A1,A2. A significant difference was detected when comparing the serum concentration at 10% with the concentration at 5% (χ2 = 3.875, df = 1, p = 0.049, <0.05) (Figure 6A1). The number of OEC in Group A was greater than the number in the Group B, and the difference was statistically significant (χ2 = 166.279, df = 1, p < 0.01) (Figure 6A2). No statistically significant difference was observed regarding the different orders in which the serum was applied (χ2 = 0.007, df = 1, p = 0.931, >0.05) (Figure 6A2). The concentration of NT-3 in the culture supernatant was shown in Figure 7. The average purity of OEC in each group is shown in Table 1. (Supplemental Table S1).


Effects of different sera conditions on olfactory ensheathing cells in vitro.

Lu M, Dong J, Lu T, Lv H, Yang P, Cheng Z, Li J, Liang B, Xu J, Li H, He X - Int J Mol Sci (2014)

Immunofluorescence staining of OEC cultured in DF-12 medium containing rat serum (RS) (concentrations: 5%, 10%, 15%, 20%) for 7 days. The concentrations of RS are listed on the left. OEC nuclei stained with DAPI are presented in the left column, P75 of OEC stained with Cy3 are presented in the middle column, and the right column is the merged nuclei and P75. Scale bar is 200 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307254&req=5

ijms-16-00420-f002: Immunofluorescence staining of OEC cultured in DF-12 medium containing rat serum (RS) (concentrations: 5%, 10%, 15%, 20%) for 7 days. The concentrations of RS are listed on the left. OEC nuclei stained with DAPI are presented in the left column, P75 of OEC stained with Cy3 are presented in the middle column, and the right column is the merged nuclei and P75. Scale bar is 200 μm.
Mentions: After P75 immunofluorescence staining, an increased number of cells in the visual field were observed (Figure 1, Figure 2, Figure 3 and Figure 4). The immunofluorescence of OEC from Group E was shown in Figure 5. The number of OEC per unit area was shown in Figure 6A1,A2. A significant difference was detected when comparing the serum concentration at 10% with the concentration at 5% (χ2 = 3.875, df = 1, p = 0.049, <0.05) (Figure 6A1). The number of OEC in Group A was greater than the number in the Group B, and the difference was statistically significant (χ2 = 166.279, df = 1, p < 0.01) (Figure 6A2). No statistically significant difference was observed regarding the different orders in which the serum was applied (χ2 = 0.007, df = 1, p = 0.931, >0.05) (Figure 6A2). The concentration of NT-3 in the culture supernatant was shown in Figure 7. The average purity of OEC in each group is shown in Table 1. (Supplemental Table S1).

Bottom Line: Compared with FBS, RS reduced OEC proliferation in relation to OEC counts (χ2 = 166.279, df = 1, p < 0.01), the optical density (OD) value in the MTT assay (χ2 = 34.730, df = 1, p < 0.01), and NT-3 concentration in the supernatant (χ2 = 242.997, df = 1, p < 0.01).There was statistically significant difference in NT-3 gene expression among different groups when the serum concentration was 15% (χ2 = 64.347, df = 1, p < 0.01).In conclusion, different serum conditions may be responsible for the variations in OEC proliferation and function.

View Article: PubMed Central - PubMed

Affiliation: Second Department of Orthopedics, Second Affiliated Hospital of Xi'an Jiaotong University, 157 West Five Road, Xincheng District, Xi'an 710004, Shaanxi, China. lumeng6020@163.com.

ABSTRACT
Transplantation of olfactory ensheathing cells (OEC) is a promising therapy in spinal cord injury (SCI) treatment. However, the therapeutic efficacy of this method is unstable due to unknown reasons. Considering the alterations in the culture environment that occur during OEC preparation for transplantation, we hypothesize that these changes may cause variations in the curative effects of this method. In this study, we compared OEC cultured in medium containing different types and concentrations of serum. After purification and passage, the OEC were cultured for 7 days in different media containing 5%, 10%, 15% or 20% fetal bovine serum (FBS) or rat serum (RS), or the cells were cultured in FBS-containing medium first, followed by medium containing RS. In another group, the OEC were first cultured in 10% FBS for 3 days and then cultured with rat spinal cord explants with 10% RS for another 4 days. An MTT assay and P75 neurotrophin receptor immunofluorescence staining were used to examine cell viability and OEC numbers, respectively. The concentration of neurotrophin-3 (NT-3), which is secreted by OEC into the culture supernatant, was detected using the enzyme-linked immunosorbent assay (ELISA). RT-PCR was applied to investigate the NT-3 gene expression in OEC according to different groups. Compared with FBS, RS reduced OEC proliferation in relation to OEC counts (χ2 = 166.279, df = 1, p < 0.01), the optical density (OD) value in the MTT assay (χ2 = 34.730, df = 1, p < 0.01), and NT-3 concentration in the supernatant (χ2 = 242.997, df = 1, p < 0.01). OEC cultured with spinal cord explants secreted less NT-3 than OEC cultured alone (F = 9.611, df = 5.139, p < 0.01). Meanwhile, the order of application of different sera was not influential. There was statistically significant difference in NT-3 gene expression among different groups when the serum concentration was 15% (χ2 = 64.347, df = 1, p < 0.01). In conclusion, different serum conditions may be responsible for the variations in OEC proliferation and function.

Show MeSH
Related in: MedlinePlus