Cell culture compositions with antioxidants and methods for polypeptide production
A technology of reagents and reagent kits, applied in the field of reagents and reagents for maintaining the viability of cancer cells in surgically removed tissues
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[0080] The preparation time used to prepare the reagents varies due to batch size, temperature, and the like. Typically, about one hour is required to complete the formulation of the cell viability reagent. The reagents can be stored at ambient conditions for up to 12 months prior to use in tissue samples. The reagents can also be frozen for longer storage.
[0081] Once the tissue sample has been added to the cell viability reagent, the tissue is stabilized and maintained at a temperature of 30°C for up to 72 hours of viable gene expression components. Typically, the cells are viable for about 30 hours to about 50 hours, and more particularly about two days. This time frame is sufficient for detailed gene expression analysis of biopsies (tissue samples) that can give important insights into the type and progression of diseases such as cancer.
[0082] refer to figure 2 , which is a flow chart illustrating an embodiment of a method for generating a cell viability reagent ...
Embodiment I
[0112] This example is an illustration of one embodiment of a method of preparing a reagent for maintaining cell viability in a tissue sample. Figure 3A and 3B is a picture of a preparation list listing the components of the reagents and instructions on how to prepare the cell viability reagent for tissue biopsy samples. In this example, one liter of reagent was prepared. With the exception of the initially measured purified water (50ml), the ingredients on the formulation list are listed in the order in which they will be added. A chaotropic agent, 8.1 gm of sodium thiocyanate, was added to the water to give a final concentration of sodium thiocyanate of 10% wv. Mix the sodium thiocyanate until the solution is clear. After addition of the chaotropic agent, the chelating agent EDTA at a stock concentration of 0.1 M was added to the solution. Add 100 ml of 0.1 MEDTA to give a final concentration of 0.01 MEDTA in the reagent. Mix the EDTA until the solution is homogeneous....
Embodiment II
[0116] Example II is a study of the effect of one embodiment of a tissue cell preservation reagent on preservation of LNCa-FGC cells and PC-3 cells. The results of preserving LNCa-FGC cells in TAG-1 reagent compared to standard cell resuspension solutions are shown in Figure 4 middle. The results of preserving PC-3 cells in TAG-1 reagent compared to standard cell resuspension solutions are shown in Figure 5 middle.
[0117] Human prostate cell lines DU145, PC-3 and LNCaP-FGC were purchased from the American Type Collection (Manassas Virginia, USA). DU145 cells were cultured in DMEM medium supplemented with 10% PBS plus penicillin (100 units / ml) and streptomycin (100 ul / ml). PC-3 and LNCaP-FGC cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin (100 units / ml) and streptomycin (100 ul / ml). Cells were grown to approximately 70% confluence in tissue culture plates. Cells are then harvested by traditional methods and resuspend...
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