Both cultured and in vitro transformed cells from tumors exhibit abnormalities in growth control, such as higher saturation densities, formation of colonies in agar, and growth of homogeneous cells into confluent monolayers. These cell lines show a lower dependence on serum concentration and growth factors and generally have a higher rate of clone formation. It is also hypothesized that a certain degree of autonomous growth is achieved due to overexpression of oncogenes or absence of suppressor genes. Growth control is usually autocrine, e.g., the cell secretes mitogens. For this reason, cells usually have receptors or express them, or their signaling is in a permanently activated or unregulated phase. Although immortality does not imply a loss of growth control, many cells are prone to progress from immortality to abnormal growth, mostly due to genetic instability of the intrinsic immortality genotype. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition
Operation method
Density-limiting experiments on cell proliferation
Principle
Cells were cultured in unrestricted medium and grown to pack and density. The percentage of 3 H-thymidine deoxyriboside-labeled cells was determined by radioautography.
Materials and Instruments
Cells for Passaging Move I. Materials Caveat 1. H3-Thymidine deoxynucleoside must be handled with great care because, although it emits only low-energy beta rays, it is capable of doping DNA and causing radiation damage. Therefore, gloves must be worn during handling, and H3-Thymidine deoxynucleoside should not be handled in a vertical laminar flow hood, but in a biohazard or cytotoxic stock laminar flow hood, and discarded liquids and solids should be disposed of in accordance with the German regulations for the handling of radioisotopes. 2. Transfer coverslips to 24-well culture plates for trypsinization to facilitate radioautography. Cells can be fixed in suspension and prepared for staining by dropping onto slides (see Scheme 16. 7， no hypotonic treatment is required), or cells can be centrifuged onto slides with a flaker (see Scheme 16.4) or adsorbed onto filter paper by vacuum filtration (see Scheme 16.5). For more product details, please visit Aladdin Scientific website.
Growth medium Maintenance medium D-PBSA Trypsin
24-well culture plate Petri dish
aseptic
1. Prepare cells for passaging
2. Growth medium
3. Maintenance medium (without serum or growth factors) containing 37 KBq/ml (1uCi/ml) of 3 H-thymidine deoxyriboside, 74 GBq/mmol (2Ci/mmol)
4. D-PBSA
5. 25% Trypsin
6. 24-well plate including 13 mm diameter coverslips
7. 9 cm diameter Petri dishes (bacteriological grade, one coverslip and one petri dish)
Procedure
1. Treat the cells with trypsin and inoculate them into 24-well plates at 1×105 cells/ml, 1 ml/well, and place a 13 mm diameter coverslip in each well.
2. Incubate the cells in a humidified CO2 incubator for 1~3d.
3. Transfer coverslips to 9 cm bacterial-grade petri dishes, each containing 20 ml of culture solution, and place the dishes in a CO2 incubator.
4. Continue incubation and change the culture solution every 2d when the cells on the coverslips are confluent.
5. Every 3-4 days, trypsin digest the cells on 2 coverslips and count them. When the cells become dense on the coverslips, 200-500 U/ml of crude collagenase must be added to the trypsin in order to completely separate the cells for counting.
6. When the cells stop growing, i.e. two counts do not show a significant increase, 2.0 ml of 37 KBq/ml (1.0 uCi/ml) of 3 H-thymidine deoxynucleoside is added to reach a final concentration of 74 GBq/mmol (2 uCi/mmol), and the cells are continued to be incubated for 24 hours.