Question

1. Describe the requirements and caveats to culturing animal cells; what nutrients and materials/substrate are required; some cell types need special growth substrates (behave differently in suspension – unlike in tissue)

2.Explain a primary culture vs. a cell line and the striking difference that tends to permit cell lines to be isolated

3. Understand how chemical screens are utilized

Answer 1

*****************************************ANSWER TO 1)****************************************************

Animal cells, unlike microbial culture, requires a variety of nutritional factors like amino acids, vitamins, hormones, growth factors. Microbes can synthesize several amino acids on their own but animal cells can't. Hence it must be supplemented with the media. To fulfill this we add serum to the media, eg-FBS (Foetal Bovine Serum), which contains several of the above-listed substances. Generally, antibiotics are also added to prevent microbial contamination. a CO₂ - Bicarbonate based buffer like HEPES, is also used to ensure proper pH. The temperature is 37 degree celsius. Oxygen pressure is also maintained

Cells may be loosely divided into two main types:

1- Suspension cell culture (Anchorage-independent)

• derived from cells which can divide and survive without being attached to a substrate,
• e.g. cells of hemopoietic lineage
• Can be maintained in culture vessels that are not tissue-culture treated,
• requires agitation for adequate gas exchange
• Easier to passage

2 Adherent cell culture (Anchorage-dependent)

• must adhere to a surface to survive
• Form monolayers
• e.g. cells derived from different tissue (breast, liver)
• Growth is limited by surface area
• Will cease proliferating once they become confluent (completely cover the surface of cell culture vessel)
• Cells are dissociated enzymatically or mechanically from surface

**********************************************ANSWER TO 2)*******************************

When cells are surgically removed from an organism and placed into a suitable culture environment they will attach, divide and grow. Most of the primary culture cells have a finite lifespan of 5-10 divisions in vitro. Due to their limited lifespan, one cannot do long-term experiments with these cells.Primary cells are considered by many researchers to be more physiologically similar to in vivo cells

If the cells in a cell strain undergo a transformation process (spontaneous or induced changes in karyotype, morphology or growth properties) that makes them "immortal“ (able to divide indefinitely) they are called a cell line. It is not known how a diploid cell strain becomes a cell line, although this event may be mimicked by infection with oncogenic viruses or by exposure to chemical carcinogens. Cell Lines often have abnormal chromosome numbers and maybe tumorigenic when inoculated into susceptible animals. Cell lines that have been derived from tumors often do not exhibit contact-inhibition (inhibition of growth under crowded conditions), but rather continue to pile-up

**********************************************ANSWER TO 3)*******************************

Chemical screens are compounds that help us quantify a certain biomolecule or activity in cell cultures. These days they are actively used in research. They might be fluorescent in nature that can allow us to detect a certain process or function. Eg. Chemical Screens for Protection Against Cisplatin-Induced Ototoxicity was developed by St. Jude Children's Research Hospital. Several chemical screens have been developed for Zebrafish at Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School by Peterson and Fishman.