The effect of TPA, a potent tumor promoter, on SSV-NIH3T3 cells in serum-free medium was investigated. TPA stimulated DNA synthesis of SSV-NIH3T3 cells on the third day of culture in SFM. In SDS-PAGF of medium conditi...The effect of TPA, a potent tumor promoter, on SSV-NIH3T3 cells in serum-free medium was investigated. TPA stimulated DNA synthesis of SSV-NIH3T3 cells on the third day of culture in SFM. In SDS-PAGF of medium conditioned by TPA-treated SSV-NIH3T3 cells (in SFM+TPA), the amounts of four proteins of 31.0 Kd, 28.5 Kd, 25.5 Kd and 13.5 Kd strikingly increased over that of non-TPA-treated counterpart (in SFM). The PDGF-like activity was also detected in CM of SFM+TPA. When insulin and EGF were drown off the SFM+TPA (SFM-Ins-EGF+TPA), TPA lost its ability to stimulate DNA synthesis of SSV-NIH3T3 cells on the third day and SDS-PAGE of the conditioned medium showed that the amounts of the four proteins noted above grately reduced. However, cells in SFM-Ins-EGF+TPA were in almost the same growth condition as cells in complete SFM+TPA on the third day of culture. Results were discussed in the paper.展开更多
A new method of long-term culture of cytotrophoblast cells in serum-free medium has been developed. Cytotrophoblast cells were isolated with cold trypsin and purified by unit gravity sedimentation through BSA density ...A new method of long-term culture of cytotrophoblast cells in serum-free medium has been developed. Cytotrophoblast cells were isolated with cold trypsin and purified by unit gravity sedimentation through BSA density gradients. The cells were cultured in the FD medium with supplement of EGF, insulin, transferrin and sodium selenite. They could survive over three weeks. The results showed that both EGF and insulin stimulated hCG and progesterone secretion and that sodium selenite elevated hCG output but not progesterone secretion. Transferrin produced synergistic effect with EGF and insulin on hCG and progesterone secretion but it was ineffective when used alone. This study demonstrates that the four growth factors mentioned above are essential for the survival of cytotrophoblast cells in vitro. It is therefore suggested that EGF, insulin and selenium may possibly be involved in the regulation of hCG and progesterone secretion in the human placenta.展开更多
Background Keratinoyte serum-free medium （K-SFM） is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer ...Background Keratinoyte serum-free medium （K-SFM） is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells （CSCs） using K-SFM. Methods＇ A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum （FCS） was used as a control. CSCs were identified with flow cytometry using CD44＋/CD24-as molecular markers. The expression of a variety of CSC markers （Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin） was analyzed with real-time PCR. Results Much higher percentage of CSCs was achieved with K-SFM： 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin. Conclusion K-SFM is an optimal culture medium to maintain and to enrich breast CSCs.展开更多
To compare the differences between proliferation and cytotoxicity of adherent natural killer （A-NK） cells cultured with serum-free medium AIMV and standard serum-containing medium in vitro, and also observe the assi...To compare the differences between proliferation and cytotoxicity of adherent natural killer （A-NK） cells cultured with serum-free medium AIMV and standard serum-containing medium in vitro, and also observe the assisting effect of IL-12 on the activation and the morphology character of IL-2-treated A-NK cells, cellular proliferation was evaluated by MTT method in vitro. The morphology of the target cells killed by A-NK cells was observed through electroscope. All of the A-NK cells cultured in serum-free medium AIMV could rapidly proliferate and keep high cytotoxicity compared with that in standard serum-containing medium. A-NK cells activated by both moderate-dose IL-2 and IL-12 were superior to the high-dose IL-2-treated A-NK cells. These data indicated that serum-free medium AIMV could replace standard serum-containing medium for culturing A-NK cells, and moderate-dose IL-2 and IL-12 could reduce side effects caused by high-dose IL-2. The study provided a new experimental basis for experimental and clinical preparation of A-NK cells. Cellular ＆ Molecular Immunology.展开更多
The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate ...The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 10<sup>7</sup> FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.展开更多
文摘The effect of TPA, a potent tumor promoter, on SSV-NIH3T3 cells in serum-free medium was investigated. TPA stimulated DNA synthesis of SSV-NIH3T3 cells on the third day of culture in SFM. In SDS-PAGF of medium conditioned by TPA-treated SSV-NIH3T3 cells (in SFM+TPA), the amounts of four proteins of 31.0 Kd, 28.5 Kd, 25.5 Kd and 13.5 Kd strikingly increased over that of non-TPA-treated counterpart (in SFM). The PDGF-like activity was also detected in CM of SFM+TPA. When insulin and EGF were drown off the SFM+TPA (SFM-Ins-EGF+TPA), TPA lost its ability to stimulate DNA synthesis of SSV-NIH3T3 cells on the third day and SDS-PAGE of the conditioned medium showed that the amounts of the four proteins noted above grately reduced. However, cells in SFM-Ins-EGF+TPA were in almost the same growth condition as cells in complete SFM+TPA on the third day of culture. Results were discussed in the paper.
文摘A new method of long-term culture of cytotrophoblast cells in serum-free medium has been developed. Cytotrophoblast cells were isolated with cold trypsin and purified by unit gravity sedimentation through BSA density gradients. The cells were cultured in the FD medium with supplement of EGF, insulin, transferrin and sodium selenite. They could survive over three weeks. The results showed that both EGF and insulin stimulated hCG and progesterone secretion and that sodium selenite elevated hCG output but not progesterone secretion. Transferrin produced synergistic effect with EGF and insulin on hCG and progesterone secretion but it was ineffective when used alone. This study demonstrates that the four growth factors mentioned above are essential for the survival of cytotrophoblast cells in vitro. It is therefore suggested that EGF, insulin and selenium may possibly be involved in the regulation of hCG and progesterone secretion in the human placenta.
基金This work was supported by a grant from the National Natural Science Foundation of China （No. 81001102）.
文摘Background Keratinoyte serum-free medium （K-SFM） is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells （CSCs） using K-SFM. Methods＇ A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum （FCS） was used as a control. CSCs were identified with flow cytometry using CD44＋/CD24-as molecular markers. The expression of a variety of CSC markers （Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin） was analyzed with real-time PCR. Results Much higher percentage of CSCs was achieved with K-SFM： 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin. Conclusion K-SFM is an optimal culture medium to maintain and to enrich breast CSCs.
文摘To compare the differences between proliferation and cytotoxicity of adherent natural killer （A-NK） cells cultured with serum-free medium AIMV and standard serum-containing medium in vitro, and also observe the assisting effect of IL-12 on the activation and the morphology character of IL-2-treated A-NK cells, cellular proliferation was evaluated by MTT method in vitro. The morphology of the target cells killed by A-NK cells was observed through electroscope. All of the A-NK cells cultured in serum-free medium AIMV could rapidly proliferate and keep high cytotoxicity compared with that in standard serum-containing medium. A-NK cells activated by both moderate-dose IL-2 and IL-12 were superior to the high-dose IL-2-treated A-NK cells. These data indicated that serum-free medium AIMV could replace standard serum-containing medium for culturing A-NK cells, and moderate-dose IL-2 and IL-12 could reduce side effects caused by high-dose IL-2. The study provided a new experimental basis for experimental and clinical preparation of A-NK cells. Cellular ＆ Molecular Immunology.
文摘The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 10<sup>7</sup> FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.
