BISC220/S10: Mod 3 Lab 9

== Cell Culture Techniques == Tissue culture (cell culture) was introduced at the beginning of the 20th century as a method for studying animal cells. The term tissue culture is derived from the original technique that used fragments of tissue surrounded by a nutrient medium. At first cell cultures were mainly the result of migration of cells from a tissue fragment into the surrounding medium, but by the 1950's primary cell cultures could be created from dispersed cells in liquid nutrient medium. Passaging of a primary cell cultures creates a cell line. Cell lines made from normal cells cannot be maintained forever, but immortalized cell lines derived from cancer cells or otherwise abnormal can often be passaged indefinitely.

Cell culture is an important technique in the study of cellular physiology. Cell culture facilitates the investigation of cell structure, reproduction, protein synthesis, energy metabolism, cell-cell interactions and environmental influences on growth. One of the advantages of using cell culture is that the physiochemical environment (pH, temperature, osmotic pressure, O₂ and CO₂ tension) can be precisely controlled. Growing cells in culture also enables a specific type of cell to be exposed directly to a test agent without interference from excretion or metabolic breakdown in a living animal.

A disadvantage associated with cell culture is that all manipulations must be performed under strict aseptic conditions to prevent contamination. A contaminated culture is useless. The validity of the cultured cell as an accurate model of in vivo physiologic function may also be questioned, since the 3-dimensional geometry of the cells in the original tissue is not strictly maintained and some of the cell lines are of metabolically abnormal cancer cells.

Cells in cell culture are suspended in a nutrient liquid medium containing amino acids, vitamins, salts, glucose and blood serum to support the growth of the cells. Serum is a necessary additive to cell culture media because it supplies the cells with many growth factors that they need to proliferate and survive in vitro. The culture is then incubated at 37°C in an environment of 95% air and 5% CO₂. Under these conditions, some of the tissue cells will create a monolayer that adheres to the culture flask and forms the basis of the primary culture.

Many cultured cells adhere to a substrate surface in the culture flask. The most common substrate materials are glass, or plastic that has been treated with chemicals or radiation to make the surface more hydrophilic so the cells will readily adhere. When cells occupy all of the substrate surface area, a state of confluence is reached. Once a confluent monolayer of cells has formed in the culture vessel, normal cells stop dividing. At this time the cells need to be supplied with fresh medium or sub-cultured to reduce the density of cells in the culture flask so growth can resume. After the first sub-culture or passage, the primary culture becomes a cell line. With each subculture, faster growing cells will soon predominate as the slower growing cells are diluted out of the population. This results in a relatively uniform population of cells available for experimentation. The number of times a cell line can be successfully subcultured depends on the type of cell. Many normal cells may only be subcultured a limited number of times, while most cancer cells can be sub-cultured indefinitely.

In the lab today, each group will be given a culture of 3T3 fibroblast cells derived from mouse embryos. Each group member will aseptically create a subculture. The cells will be supplied at or near confluence, so the rate of growth of the cells has ceased and subculture is necessary. Sub-culture of the fibroblasts involves removal of the medium from the flask, exposure of the cells to the protease, trypsin, to disassociate them from the substrate surface, and dispersal of the cells at the proper density in fresh medium. You should be able to prepare 2-3 cell culture flasks seeded at 2.4 x 10⁴ cells/ml from the original culture.

Materials Provided for Cell Culture:

1. Dulbecco's modified Eagle Medium containing salts, amino acids, vitamins and glucose, with 10% calf serum (sterile)
2. Phosphate buffered saline, pH=7.4 (sterile) 137mM NaCl, 2.7 mM KCl, 10mM Na₂HPO₄, 2 mM KH₂PO₄
3. 0.25% Trypsin in sterile PBS
4. Sterile pipettes: 1 ml, 5 ml and 10 ml
5. 0.1% Trypan Blue stain
6. Hemocytometer & coverslip
7. Microscope
8. Tissue culture flasks, 25 cm²
9. Culture of 3T3 fibroblasts

Protocol