Methods and reagents for maintaining the viability of cancer cells in surgically removed tissue
A reagent, cell technology, used in tissue culture, animal cells, biochemical equipment and methods, etc.
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[0080] Preparation times for preparing the reagents vary due to batch size, temperature, and the like. Typically, it takes about one hour to complete the preparation of the cell viability reagent. The reagents can be stored under 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 maintains viable gene expression components at a temperature of 30°C for up to 72 hours. Typically, the cells are viable for about 30 hours to about 50 hours, and more specifically about two days. This time frame is sufficient for detailed gene expression analysis of biopsies (tissue samples), which can give important insights into the type and progression of diseases such as cancer.
[0082] refer to figure 2 , which is a flow diagram illustrating an embodiment of a method for producing a cell viability agent according ...
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 with 3B is a picture of a recipe list listing the components of the reagent and instructions on how to prepare the cell viability reagent for use in tissue biopsy samples. In this example, one liter of reagent was prepared. Except for the initial measure of 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. Sodium thiocyanate was mixed until the solution was clear. After addition of the chaotropic agent, 0.1 M stock concentration of the chelating agent EDTA was added to the solution. 100 ml of 0.1 M EDTA was added to give a final concentration of 0.01 M EDTA in the reagent. Mix EDTA until the solution is homogeneous. The n...
Embodiment II
[0116] Example II is a study of the effect of an embodiment of the 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 solution are shown in Figure 4 middle. The results of preserving PC-3 cells in TAG-1 reagent compared to standard cell resuspension solution are shown in Figure 5 middle.
[0117] Human prostate cell lines DU145, PC-3 and LNCaP-FGC were purchased from American Type Culture Collection (Manassas Virginia, USA). DU145 cells were cultured in DMEM medium supplemented with 10% PBS plus penicillin (100 units / ml) and streptomycin (100ul / ml). PC-3 and LNCaP-FGC cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin (100 units / ml) and streptomycin (100 ul / ml). Cells were grown to approximately 70% confluency in tissue culture plates. Cells are then harvested by conventional means and resuspe...
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