Cell therapeutic agent for cancer treatment and combination therapy with same
A cancer treatment and cell technology, applied in the direction of genetically modified cells, cell culture active agents, animal cells, etc., can solve the problem of not doing too much research, and achieve the effect of maximizing the effect and excellent tumor inhibition effect.
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preparation Embodiment 1
[0073] Preparation Example 1: Preparation of Mesenchymal Stem Cells Expressing Cytosine Deaminase (CD)
[0074] 1.1 Isolation and culture of mesenchymal stem cells
[0075]4 ml of human bone marrow donated after IRB examination at Ajou University Medical Center was overlaid with 4 ml of HISTOPAQUE 1077 (Sigma-Aldrich) in a sterile 15 ml tube and centrifuged at 400 x g for 30 at room temperature. minute. After centrifugation, 0.5 ml of the intermediate buffy coat was carefully collected using a Pasteur pipette and, after centrifugation at 250 x g for 10 minutes, transferred to a test tube containing 10 ml of sterile phosphate buffered saline (pH 7.4) , discard the supernatant, and add 10 ml of phosphate buffered saline, and gently suspend, then centrifuge at 250 x g for 10 min. This operation was repeated twice, and the final pellet was added to DMEM medium (Gibco) supplemented with 10% FBS (like standard fetal bovine serum (Hyclone)) and dispersed in 100 mm animal cell cultu...
Embodiment 1
[0082] Suicide effect and bystander effect of embodiment 1.MSC / CD
[0083] 1.1 Confirm the suicide effect
[0084] Cytosine deaminase (CD) is a suicide gene. MSC / CD, which are mesenchymal stem cells expressing cytosine deaminase (CD), can convert the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and can have figure 1 Suicide effect (cells killing themselves) and bystander effect (killing of surrounding cells) are shown. Therefore, the suicide effect and bystander effect of MSC / CD prepared in specification 1.3 were eliminated. First, 10,000 MSC / CD or mesenchymal stem cells (MSC) were cultured in a 12-well plate. From the next day, cells were treated with prodrug 5-FC at a concentration of 0-1,000 μM, and replaced every two days with new medium containing the drug. Change medium to cell culture medium containing 0.5 mg / ml MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) A base capable of measuring live cells on day 6 after treat...
preparation Embodiment 2
[0090] Preparation Example 2. Preparation of "I cell" and "F cell"
[0091] In the case of cell therapeutics, the effect can vary under various conditions according to changes in the state of the cells. Therefore, in order to confirm whether cell ability changes depending on the state of MSC / CD, cells in a state of thawing immediately after freezing (hereinafter, referred to as F cells (frozen cells)), and cells in a state of collecting from cells immediately during culture (hereinafter, referred to as F cells) were prepared. for I cells (cells collected immediately)).
[0092] To prepare F cells and I cells separately, MSC / CD was cultured, and then 2×10 6 Disperse each cell in 1ml freezing medium containing 2%-10% (2%, 5%, 10%) DMSO or 2×10 6 The cells were dispersed in 1 ml of CryostorCS10 (BioLife Solutions). Cells were frozen and stored in liquid nitrogen containers. First, to obtain "I cells" of MSC / CD, cells from passage 5 were lysed at 37°C and placed in 9 ml of cul...
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