5637 human bladder cancer cells

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Cell line/strain:

1. Cell Line: A primary culture cell line that has been successfully passaged consists of a lineage of cells that originally existed in the primary culture.

2. Cell Strain: A cell line obtained from primary cultures or cell lines through selection or clone formation methods, which has specific properties or markers, is called a cell line. The special properties or markers of the cell line must be present throughout the entire culture period. If the number of passages cannot continue or is limited, it is called a finite cell line; If it can be continuously passaged, it is called a continuous cell line. For human tumor cells, those that have been cultured in vitro for more than six months, have stable growth, and have been continuously passaged can be called continuous strains or lines.

（1） First generation cultivation

Primary culture, also known as primary culture, refers to the first culture of cells, tissues, and organs directly taken from the body. Once subcultured, cells are no longer referred to as primary culture but rather as cell lines.

（2） Cell line

The cells after the first passage of the primary culture are called cell lines. If the survival period of a cell line is limited, it is called a Finite Cell Line; A cell line that has acquired unlimited reproductive ability and can continue to survive is called a continuous cell line or an infinite cell line. Most infinite cell lines have undergone aneuploidy, with an aneuploid karyotype, and some may have become malignant cells, so they are essentially transformed cell lines. Infinite cell lines may only have immortality (or immortality), but still retain contact inhibition and no carcinogenicity from allogeneic inoculation; Some not only have immortality, but allogeneic vaccination also has tumorigenicity, indicating malignancy. These two types of infinite cell lines with different properties are often not strictly applied to these terms in domestic and foreign literature. For conceptual clarity, this book uses infinite cell lines with malignancy! Malignant transformed cell line! The word ± may be more appropriate. For cell lines that only have immortality and no malignancy, infinite cell lines or transformed cell lines can be used. The currently circulating cell lines such as NIH3T3, Rat-1, 10T1/2, etc. belong to this category.

Cell culture operation:

1) Resuscitate cells: Quickly shake and thaw a cryovial containing 1 mL of cell suspension in a 37 ℃ water bath, and mix well with 4 mL of culture medium. Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 1-2 mL of culture medium, and blow well. Then add all cell suspensions to a culture bottle containing an appropriate amount of culture medium and culture overnight (or add the cell suspensions to a 6 cm dish, add about 4 mL of culture medium, and culture overnight). The next day, change the solution and check the cell density.

2) Cell passage: If the cell density reaches 80% -90%, passage culture can be carried out. a、 Discard the culture supernatant and rinse the cells 1-2 times with PBS that does not contain calcium or magnesium ions.

b、 Add 1 mL of digestion solution (0.25% Trypsin 0.53mM EDTA) to the culture bottle to allow the digestion solution to infiltrate all cells. Discard the digestion solution and place the culture bottle in a 37 ℃ incubator for 1-3 minutes (depending on the digestion of the cells). Then observe the digestion of the cells under a microscope. If most of the cells become round and fall off, quickly take them back to the operating table, tap the culture bottle a few times, and add 2-3 ml of culture medium to terminate digestion. Gently mix well and transfer to a sterile centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 1-2 mL of culture medium, and blow well. .

c、 Divide the cell suspension into a new dish or bottle containing 8 mL of culture medium at a ratio of 1:2, and place it in a culture incubator for cultivation.

3) Cell cryopreservation: When the cells are in good growth condition, cell cryopreservation can be performed. Taking T25 bottles as an example below;

a、 Collect cells, transfer them into sterile centrifuge tubes, centrifuge at 1000 rpm for 4 minutes, discard the supernatant, wash with PBS, discard PBS, and perform cell counting.

b、 Add serum-free cell cryopreservation solution according to the number of cells, make the cell density 5 × 106~1 × 107/mL, gently mix well, and freeze 1mL of cell suspension in each cryopreservation tube, paying attention to labeling the tubes properly.

c、 Place the cryovial in a -80 ℃ freezer and transfer it to liquid nitrogen for storage after 24 hours. Record the location of the freezer for future retrieval.

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