Ovarian cancer stem cell vaccine and preparation method thereof
A technology of ovarian cancer cells and stem cells, which is applied in the fields of molecular biology and genetic engineering, can solve problems such as inability to achieve effective remission and treatment effects
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Embodiment 1
[0046] Example 1 Preparation of Ovarian Cancer Cells
[0047] a. After obtaining the tumor tissue aseptically, wash the tumor tissue with PBS containing double antibody to remove necrotic tissue, connective tissue, blood vessels, fat, etc., and cut the tumor tissue into 1mm with scissors 3 Add collagenase and DNase, digest and enzymatically digest, filter and prepare single cell suspension;
[0048] b. Use immunomagnetic beads to isolate fibroblasts;
[0049] c. After the negative selection liquid is placed in the culture flask, after the cells adhere to the wall, remove the suspended cells, replace the fresh medium, and subculture.
Embodiment 2
[0050] Example 2 Ovarian Cancer Stem Cell CD117 + CD44 + Preparation
[0051] Cell culture and immunomagnetic bead sorting
[0052] 1. Culture the human EOCSKOV3 cells with 10% fetal bovine serum RPMI1640 as a medium, and culture them at 37°C and 5% CO2 saturated humidity. After the cells grow to a certain density, trypsin digestion and blow off, then continue subculture.
[0053] 2. CD44+SKOV3 cell immunomagnetic bead sorting (magnetic activated cell sorting, MACS)
[0054] (1) Take 300μl of MACS buffer to suspend SKOV3 cells;
[0055] (2) Every 1×10 7 ~1×10 8 Cells, add 100μl FcR blocking agent, mix well and incubate at 4℃ for 30min;
[0056] (3) Every 1×10 7 ~1×10 8 Cells, add 100μl CD44+ magnetic bead labeled direct-labeled monoclonal antibody, mix well and incubate at 4°C for 30min, then add PBS to 1mL, centrifuge at 1000rpm for 8min to collect the cell pellet;
[0057] (4) Add PBS (containing EDTA) solution to 1 mL, centrifuge at 1000 rpm for 8 min, wash the cells once, discard the ...
Embodiment 3
[0071] Example 3 CD117+CD44+SKOV3 cell morphology observation and surface marker detection
[0072] Under the inverted microscope, continuously and dynamically observe the cell growth status, and take 1×10 CD117+CD44+SKOV3 cells cultured in vitro 6 Cells were labeled with CD117-PE and CD44-FITC monoclonal antibodies respectively, and incubated in a 37°C, saturated humidity, 5% CO2 incubator for 30 minutes, PBS washed to remove excess antibodies, FCM detected CD117+CD44+SKOV3 surface markers.
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