Figure 1.
CD133(+) SAOS-2 cells are selected in the hydrofocusing bioreactor.
A) Trypan blue exclusion cell count of SAOS-2 cells (osteosarcoma cells) and SAOS-2 cells cultured in the HFB, CD133(+) SAOS-2 cells and CD133(+) SAOS-2 cells cultured in the HFB, and of CD133(+) SAOS-2 cells and CD133(+) SAOS-2 cells cultured in normal gravity condition. Gray bars indicate the number of cells placed in the control on day-1. Black bars indicate the number of viable cells recovered after 5 days of culture in that culturing condition. B) Bright field contrast phase image (20×) of 3-D culture (spheres) formed from SAOS-2 cells that were grown in the HFB for 5 days. Inset (top right) shows an image of CD133 immunofluorescence staining of SAOS-2 cells grown in the bioreactor for 5-days. C) Flow cytometry chart of the CD133 immunophenotype of SAOS-2 cells. An isotype antibody was used as a control. D) Trypan blue exclusion cell counts of an optimized experiment of HFB culture of SAOS-2 cells in the HFB after seven days. CD133(+) SAOS-2 cells cultured in the bioreactor were selected and proliferated 15-fold after seven days. White bar indicates the number of SAOS-2 cells seeded in the HFB. Black bar indicates the number of viable CD133(+) SAOS-2 cells recovered after 7 days of optimized culture in the bioreactor. Gray bar indicate the number of CD133(+) cells present in the parental SAOS-2 population. The data is representative of three separate experiments yielding comparable results.
Table 1.
Percentage of positivity ± standard deviation to CD133 of the various cell lines tested.
Table 2.
Comparison of CD133(+) and (−) cel counts ± standard deviation of SAOS-2 cells grown in the HFB or RCCS vessels.
Figure 2.
SAOS-2 cells die by apoptosis after 5 days of culture in the hydrofocusing bioreactor as evidenced by Annexin-V staining/Facs analysis and caspases-3 colorimetric assay.
A) Annexin-V staining of SAOS-2 cells cultured in 1-G for 3 days. The analysis allows to distinguish in the diagram between living cells (lower left quadrant), early apoptotic cells (lower right quadrant), apoptotic cells (upper right quadrant), and necrotic cells (upper left quadrant). B) Annexin-V staining of SAOS-2 cells cultured in the HFB for 3 days. C) Annexin-V staining of SAOS-2 cells cultured in 1-G for 5 days. D) Annexin-V staining of SAOS-2 cells cultured in the HFB for 5 days. E) Annexin-V staining of CD133(+) MACSorted SAOS-2 cells which were cultured in 1-G for 5 days. F) Annexin-V staining of CD133(+) MACSorted SAOS-2 cells which were cultured in the HFB for 5 days. G) Relative caspase-3 activity of SAOS-2 cells (total population) cultured for 5 days in the HFB compared to SAOS-2 cells (total population) grown in static 1G condition.
Figure 3.
Fluorescence index of various stem cell and differentiation markers examined in cells cultured in 1-G static condition, sorted by CD133 antibody or not, and after culture in the bioreactor for 5 days.
A) Diagram of the expression levels of various bio-markers. Lavender bars indicate the percentage of cells expressing basal levels of the various markers in parental SAOS-2 cells cultured in normal 1-G gravity condition (control). Purple bars indicate the percentage of cells expressing the various markers in CD133(+) MACSorted SAOS-2 cells that were isolated from parental SAOS-2 cells cultured in normal 1-G gravity condition. Yellow bars indicate the percentage of cells expressing the various markers in CD133(+) MACSorted SAOS-2 cells that were cultured in hypogravity condition. Light blue bars indicate the percentage of cells expressing the various markers in parental SAOS-2 cells that were cultured in hypogravity condition for 5-days, resulting in a population of selected and proliferated CD133(+) SAOS-2 cells. B) Immunofluorescence staining (40×) and positivity of SAOS-2 cells to CD133, Sox-9, SPARC, and CD117/c-Kit following growth in the bioreactor for 5-days.
Figure 4.
Phase contrast images of spheres formed by HFB SAOS-2 CD133(+) enriched osteosarcoma cells.
A) HFB grown SAOS-2 osteosarcoma cells (CD133+) are able to proliferate and assemble three-dimensionally as sarcosheres after 3-days of culture in the bioreactor (10× magnification). B) HFB grown SAOS-2 osteosarcoma cells (CD133+) are able to proliferate and assemble three-dimensionally as sarcosheres after 3-days of culture in the bioreactor (40× magnification). C) HFB grown SAOS-2 osteosarcoma cells (CD133+) seeded into an irradiated plastic culture dish, reconstitute the normal attached phenotype of the parental SAOS-2 cells after one week of culture (10× magnification). Arrows point at CD133(+) SAOS-2 cells showing a rounded and weakly attaching phenotype. D) HFB grown SAOS-2 osteosarcoma cells (CD133+) seeded into an irradiated plastic culture dish, reconstitute the normal attached phenotype of the parental SAOS-2 cells after one week of culture (40× magnification). Arrow points at CD133(+) SAOS-2 cells showing a rounded and weakly attaching phenotype.
Figure 5.
Phase contrast images of spheres formed by MACSorted CD133(+) SAOS-2 osteosarcoma cells in ultra low-attaching dishes.
A) MACSorted CD133(+) SAOS-2 cells assemble three-dimensionally as sarcosheres after 2 weeks of culture in ultra low-attaching dishes (20× magnification). B) Another example of MACSorted CD133(+) SAOS-2 cells forming sarco-spheres after 2 weeks of culture in ultra low-attaching dishes (10× magnification). C) SAOS-2 osteosarcoma cells deriving from three-dimensional cultures grown in ultra low-attaching dishes seeded into attaching dishes show an adherent a differentiated phenotype after 3-days. D) SAOS-2 osteosarcoma cells deriving from three-dimensional cultures grown in ultra low-attaching dishes seeded into attaching dishes show an adherent a differentiated phenotype after 5-days. E) SAOS-2 osteosarcoma cells deriving from three-dimensional cultures grown in ultra low-attaching dishes seeded into attaching dishes show an adherent and differentiated phenotype after 10 days.
Figure 6.
The SAOS-2 cell line is composed of two distinct populations.
A) Contrast phase microscopy of a soft agar assay of parental SAOS-2 cells. B) Contrast phase microscopy of a soft agar assay of HFB CD133(+) proliferated SAOS-2 cells. C) Propidium iodide flow cytometry analysis of SAOS-2 cells grown in 1-G culture sorted by their CD133 status showing two distinct populations: the CD133(+) and CD133(−) cells with a different cell cycle profile. D) Immunophenotype of SAOS-2 cells grown in 1-G versus HFB culture showing that the SAOS-2 cells grown for 5 days in simulated hypogravity contain the majority of the cells stained for Ki-67 and therefore in the S-phase of the cell cycle.
Table 3.
Average number of colonies counted ± standard deviation in 6-well dishes in a triplicate experiment.
Figure 7.
Sensitivity of SAOS-2 cells to cisplatin following growth in simulated hypogravity.
A) LD50 for cisplatin determined for the SAOS-2 cells. An LD50 of 10µg/mL for cisplatin was determined exposing the SAOS-2 cells to a 24-hours treatment using the MTT assay. Comparable results were obtained with cell count by trypan blue exclusion. B) Histogram showing the sensitivity of SAOS-2 cells to 5µg/mL of cisplatin following a 24-hours treatment, using an MTT assay. CD133(+) cells are resistant to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitive, instead. C) Histogram showing the sensitivity of SAOS-2 cells to a clinically relevant dose of 10µg/mL of cisplatin following a 24-hours treatment, using an MTT assay. CD133(+) cells are resistant to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitive, instead. D) Histogram showing the sensitivity of SAOS-2 cells of 15µg/mL of cisplatin following a 24-hours treatment, using an MTT assay. CD133(+) cells are resistant to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitive, instead.
Figure 8.
Sensitivity of SAOS-2 cells to methotrexate following growth in simulated hypogravity.
A) LD50 for methotrexate determined for the SAOS-2 cells. An LD50 of 22µg/mL for methotrexate was determined exposing the SAOS-2 cells to a 24-hours treatment to the drug, using the MTT assay. Comparable results were obtained with cell count by trypan blue exclusion. B) Histogram showing the sensitivity of SAOS-2 cells to 4µg/mL of methotrexate following a 24-hours treatment, using an MTT assay. CD133(+) cells are resistant to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitive, instead. C) Histogram showing the sensitivity of SAOS-2 cells to 11µg/mL of methotrexate following a 24-hours treatment, using an MTT assay. CD133(+) cells are resistant to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are greatly sensitive, instead. D) Histogram showing the sensitivity of SAOS-2 cells to a clinically relevant dose of 22µg/mL of methotrexate following a 24-hours treatment, using an MTT assay. CD133(+) cells are resistant to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitive, instead. E) Histogram showing the sensitivity of SAOS-2 cells to 45µg/mL of methotrexate following a 24-hours treatment, using an MTT assay. CD133(+) cells are resistant to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitive, instead.
Figure 9.
Sensitivity of SAOS-2 cells to doxorubicin following growth in simulated hypogravity.
A) LD50 for doxorubicin determined for the SAOS-2 cells. An LD50 of 0.5µg/mL for doxorubicin was determined exposing the SAOS-2 cells to a 24-hours treatment to the drug, using an MTT assay. Comparable results were obtained with cell count by trypan blue exclusion. B) Histogram showing the sensitivity of SAOS-2 cells to 0.25µg/mL of doxorubicin following a 24-hours treatment, and using an MTT assay. CD133(+) cells are sensitive to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are even more sensitive to the treatment, instead. C) Histogram showing the sensitivity of SAOS-2 cells to 0.5µg/mL of doxorubicin following a 24-hours treatment, using an MTT assay. CD133(+) cells show sensitivity to the clinically relevant dose of 0.5 µg/mL of doxorubicin, however the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are greatly sensitized to the chemotherapy treatment. D) Histogram showing the sensitivity of SAOS-2 cells to a dose of 1.1µg/mL of doxorubicin following a 24-hours treatment, and using an MTT assay. The CD133(+) cells show sensitivity to a dose of 1.1µg/mL of doxorubicin, however the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitized to the chemotherapy treatment. E) Histogram showing the sensitivity of SAOS-2 cells to 2.2µg/mL of doxorubicin following a 24-hours treatment, and using an MTT assay. CD133(+) cells are sensitive to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitive, instead.
Figure 10.
Graphic representation of the relative caspase-3 activity of SAOS-2 cells exposed to CDDP after growth stimulation in simulated hypogravity.
SAOS-2 cells grown for 5 days in the hydrofocusing bioreactor and treated with 10 µg/mL of CDDP die by apoptosis as evidenced by a caspases-3 colorimetric assay. The white bar illustrates the caspase-3 activity recovered from SAOS-2 cells grown in the HFB and then treated with CDDP for 24-hours. The black bar illustrates the caspase-3 activity recovered from SAOS-2 cells grown in static normal (1-G) condition and then treated with CDDP for 24-hours, and the grey bar indicates the basal caspases-3 activity of SAOS-2 cells grown in static 1-G condition.